Methods of treating biomass to produce oligosaccharides and related compositions

ABSTRACT

Methods of forming an ingredient for human consumption are provided herein. The methods may include isolating one or more soluble polysaccharides from a biomass, generating one or more oligosaccharides from the biomass, and combining the one or more isolated soluble polysaccharides with the generated oligosaccharides to form the ingredient. Methods of pretreating a biomass are also provided. The methods may include administering a physical pretreatment to a biomass, administering a gentle pretreatment to the physically pretreated biomass, and administering a strong pretreatment to the gently pretreated biomass. Ingredients for human consumption are also provided.

CROSS REFERENCE

This application is a continuation of International Patent ApplicationPCT/EP2020/072929, filed Aug. 14, 2020, which this application claimsthe benefit of UK Patent Application No. 1911762.1, filed Aug. 16, 2019,UK Patent Application No. 1911764.7, filed Aug. 16, 2019, and UK PatentApplication No. 2002315.6, filed Feb. 19, 2020, each of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated by inits entirety. Said ASCII copy, created Oct. 26, 2020, is named 56406 709302 ST.txt and is 97.0 kilobytes in size

BACKGROUND

Sugary foods and drinks are an important part of cultural and lifestylehabits across the world, but the sugar they contain has been linked toobesity, diabetes, poor dental health, and disruptive behavior inpeople. Because of this, consumer preferences have been shifting awayfrom sugar-containing foods, and governments are increasinglyimplementing regulation to encourage the consumption of less sugar.

As such, industry has been searching for suitable low-calorie sweetenersfor many decades to substitute for sugar in food and beverages.Unfortunately, many sugar substitutes are produced from non-naturalresources, and often offer bitter undertones or other unpleasant tastesalong with their sweetness, both of which consumers find unappealing.Moreover, while many sweeteners are able to mimic the sweetness of sugarin food and drinks, few are able to mimic the broad range of roles thatsugar plays in food, such as adding bulk, modulating texture, providingstructure, acting as a preservative, and modulating color and flavorthrough caramelization and Maillard reactions. In addition, many bulkingsweeteners that are able to mimic these physical properties of sugarhave gastrointestinal tolerance issues that limit their use to levelswell below the amount required to replace sugar in a standard Westerndiet.

Dietary fiber is an important part of a positive diet and helps maintaindigestive health and a well-regulated gut flora. Such fiber includessaccharides of varying chain lengths and types. In addition to beingfound naturally in a wide spectrum of foods, fiber can also be producedseparately and added to other foods during their manufacture.

SUMMARY

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

In some embodiments, described herein are methods of producingingredients for human consumption. A method of producing an ingredientfor human consumption may comprise (a) isolating one or more solublepolysaccharides from a biomass; (b) contacting the remaining biomasswith one or more enzymes to form one or more oligosaccharides; (c)isolating the one or more oligosaccharides; and (d) combining a portionof the one or more soluble polysaccharides from step (a) with a portionof the one or more oligosaccharides from step (c) to form theingredient.

In some cases, the method may comprise purifying the isolated one ormore soluble polysaccharides.

In some cases, the method may comprise purifying the isolated one ormore oligosaccharides.

In some cases, the method may comprise treating the biomass tosolubilize the one or more soluble polysaccharides.

In some cases, the method may comprise purifying the isolated one ormore soluble polysaccharides.

In some cases, the treating comprises a thermochemical treatment.

In some cases, the thermochemical treatment comprises at least one of ahot water treatment or a hot alkali treatment.

In some cases, the hot alkali treatment uses an alkali with a pH of from10 to 14.

In some cases, the hot alkali treatment uses at least one of sodiumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,calcium hydroxide, ammonium sulfate, ammonium hydroxide, or aqueousammonia.

In some cases, the treating may be conducted at a temperature of from30° C. to 180° C.

In some cases, the treating may be conducted for from 10 minutes to 24hours.

In some cases, the one or more soluble polysaccharides and/or the one ormore oligosaccharides are dried prior to step (d).

In some cases, the one or more soluble polysaccharides and/or the one ormore oligosaccharides are dried subsequent to step (d).

In some cases, the ingredient may be soluble in water.

In some cases, the solubility of the ingredient in water may be at least80 g of the ingredient per 100 g of water at 50° C.

In some cases, the method comprises combining the ingredient with aliquid to form a liquid ingredient.

In some cases, a viscosity of the liquid ingredient may be similar to aviscosity of corn syrup.

In some cases, a viscosity of the liquid ingredient may be similar to aviscosity of high-fructose corn syrup.

In some cases, the liquid ingredient has fewer calories per gram thancorn syrup or high-fructose corn syrup.

In some cases, the liquid ingredient has a lower glycemic index thancorn syrup or high-fructose corn syrup.

In some cases, the liquid comprises water.

In some cases, the liquid ingredient comprises at least 20% by dryweight of the at least one oligosaccharide and at least 2% by dry weightof the at least one polysaccharide.

In some cases, the liquid ingredient has a viscosity of from 5 cps to100,000 cps, 8,000 cps to 100,000 cps, 10,000 cps to 50,000 cps, or15,000 cps to 25,000 cps.

In some cases, the liquid ingredient comprises at least 2% by dry weightof xylan.

In some cases, the liquid ingredient comprises at least 2% by dry weightof mannan.

In some cases, the liquid ingredient comprises at least 2% by dry weightof a cellulose derivative.

In some cases, the liquid ingredient has a concentration ofpolysaccharides of from 0.1% to 50% w/v.

In some cases, the liquid ingredient comprises an amount ofpolysaccharide and oligosaccharide in a ratio from 1:100 to 1:1.

In some cases, the one or more soluble polysaccharides comprise at leastone of a mannan, a xylan, a mixed-linkage glucan, a lignocellulose, ahemicellulose, a cellulose derivative, a chitosan, or a xyloglucan.

In some cases, the cellulose derivative comprises at least one of acellulose acetate, a hydroxyethylcellulose, or a hydroxymethylcellulose.

In some cases, the biomass comprises at least one of a sugar canebiomass, a corn biomass, a wheat biomass, a hardwood biomass, or asoftwood biomass.

In some cases, the one or more oligosaccharides comprise at least oneof: i) a cello-oligosaccharide having a degree of polymerization (DP) offrom two to six; ii) a xylo-oligosaccharide having a DP of from two totwelve; iii) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; iv) a manno-oligosaccharide having a DP of from two to twelve;v) a mixed-linkage glucan oligosaccharide having a DP of from two tofive; vi) a xyloglucan oligosaccharide having a DP of from four totwelve; or vii) a chito-oligosaccharide having a DP of from two totwelve.

In some cases, the ingredient comprises at least two of theoligosaccharides listed in (i) to (vii).

In some cases, the ingredient comprises the at least twooligosaccharides in a ratio from 1:9 to 1:1 in relation to each other.

In some embodiments, described herein are compositions for humanconsumption.

The composition for human consumption may comprise: a solublepolysaccharide; and an oligosaccharide may comprise at least one of: (i)a cello-oligosaccharide having a degree of polymerization (DP) of fromtwo to six; (ii) a xylo-oligosaccharide having a DP of from two totwelve; (iii) a manno-oligosaccharide having a DP of from two to twelve;(iv) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; (v) a mixed-linkage glucan oligosaccharide having a DP of fromtwo to five; or (vi) a chito-oligosaccharide having a DP of from two totwelve, wherein the composition comprises less than 5% by dry weightinsoluble polysaccharides.

In some cases, the composition may be substantially free of insolublepolysaccharides.

In some cases, the composition may be soluble in water.

In some cases, the solubility of the composition in water may be atleast 80 g of the composition per 100 g of water at 50° C.

In some cases, the composition further comprises a liquid, therebyforming a liquid ingredient.

In some cases, the liquid may be water.

In some cases, the liquid ingredient comprises at least 20% by dryweight of the at least one oligosaccharide and at least 2% by dry weightof the at least one polysaccharide.

In some cases, the liquid ingredient has a viscosity of from 5 cps to100,000 cps, 8,000 cps to 100,000 cps, 10,000 cps to 50,000 cps, or15,000 cps to 25,000 cps.

In some cases, the liquid ingredient comprises at least 2% by dry weightof xylan.

In some cases, the liquid ingredient comprises at least 2% by dry weightof mannan.

In some cases, the liquid ingredient comprises at least 2% by dry weightof a cellulose derivative.

In some cases, the liquid ingredient has a concentration ofpolysaccharides of from 0.1% to 50% w/v.

In some cases, the liquid ingredient comprises an amount ofpolysaccharide and oligosaccharide in a ratio from 1:100 to 1:1.

In some cases, the one or more soluble polysaccharides comprise at leastone of a mannan, a xylan, a mixed-linkage glucan, a lignocellulose, ahemicellulose, a cellulose derivative, a chitosan, or a xyloglucan.

In some cases, the cellulose derivative comprises at least one of acellulose acetate, a hydroxyethylcellulose, or a hydroxymethylcellulose.

In some cases, the biomass comprises at least one of a sugar canebiomass, a corn biomass, a wheat biomass, a hardwood biomass, or asoftwood biomass.

In some cases, the one or more oligosaccharides comprise at least oneof: i) a cello-oligosaccharide having a degree of polymerization (DP) offrom two to six; ii) a xylo-oligosaccharide having a DP of from two totwelve; iii) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; iv) a manno-oligosaccharide having a DP of from two to twelve;v) a mixed-linkage glucan oligosaccharide having a DP of from two tofive; vi) a xyloglucan oligosaccharide having a DP of from four totwelve; or vii) a chito-oligosaccharide having a DP of from two totwelve.

In some cases, the composition comprises at least two of theoligosaccharides listed in (i) to (vii).

In some cases, the ingredient comprises the at least twooligosaccharides in a ratio from 1:9 to 1:1 in relation to each other.

In some embodiments, described herein are methods for producingingredients for human consumption. The method for producing aningredient for human consumption may comprise: (a) administering aphysical pretreatment to a biomass to reduce an average size of thebiomass; (b) administering a gentle pretreatment to the physicallypretreated biomass, the gentle pretreatment may comprise: (i) incubatingthe physically pretreated biomass in an aqueous solution to solubilizemonosaccharides and/or disaccharides from the physically pretreatedbiomass; and (ii) removing a portion of the solubilized monosaccharidesand/or disaccharides from the aqueous solution; (c) administering astrong pretreatment to the gently pretreated biomass to increase thedigestibility of the biomass; (d) contacting, in a solution orsuspension, one or more polysaccharide-cleaving enzymes and the stronglypretreated biomass to form one or more oligosaccharides; and (e)enriching the solution or suspension to increase the concentration ofthe one or more oligosaccharides to form the ingredient.

In some cases, the gentle pretreatment may be an incubation cycle.

In some cases, the strong pretreatment may be a thermochemical treatmentmay comprise incubating the gently pretreated biomass in one of anacidic solution or an alkali solution.

In some cases, the method may further comprise removing at least 25% or50% of the solubilized monosaccharides and/or disaccharides from theincubation solution at step (b)(ii).

In some cases, the strongly pretreated biomass composition after step(c) comprises less than 10% w/w monosaccharides.

In some cases, the method may further comprise purifying the one or moreoligosaccharides from the solution or suspension.

In some cases, the strongly pretreated biomass composition after step(c) comprises less than 20% w/w monosaccharides.

In some cases, the method may further comprise repeating step (b).

In some cases, the step (b) may be conducted two, three, four, or fivetimes.

In some cases, the method may further comprise repeating step (c).

In some cases, the step (c) may be conducted two, three, four, or fivetimes.

In some cases, the method may further comprise concentrating the portionof the solubilized monosaccharides and/or disaccharides removed in step(b).

In some cases, the method may further comprise discarding the portion ofthe solubilized monosaccharides and/or disaccharides removed in step(b).

In some cases, the portion of the solubilized monosaccharides and/ordisaccharides removed in step (b) may be not combined with the portionof the one or more oligosaccharides of step (e) to form the ingredient.

In some cases, the ingredient comprises less than 15% by dry weightmonosaccharides.

In some cases, the ingredient comprises less than 50% by dry weightdisaccharides.

In some cases, the ingredient may be substantially free ofmonosaccharides.

In some cases, the ingredient may be substantially free ofdisaccharides.

In some cases, the one or more oligosaccharides comprise at least oneof: i) a cello-oligosaccharide having a degree of polymerization (DP) offrom two to six; ii) a xylo-oligosaccharide having a DP of from two totwelve; iii) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; iv) a manno-oligosaccharide having a DP of from two to twelve;v) a mixed-linkage glucan oligosaccharide having a DP of from two tofive; vi) a xyloglucan oligosaccharide having a DP of from four totwelve; or vii) a chito-oligosaccharide having a DP of from two totwelve.

In some cases, the ingredient comprises at least two of theoligosaccharides listed in (i) to (vii).

In some cases, the ingredient comprises the at least twooligosaccharides in a ratio from 1:9 to 1:1 in relation to each other.

In some cases, the ingredient comprises at least one of sucrose,maltose, lactose, glucose, fructose, or galactose at less than 50% totaldry w/w of the total dry w/w of all oligosaccharides of i-vii

In some cases, the monosaccharides and/or disaccharides comprise atleast one of sucrose, maltose, lactose, glucose, fructose, or galactose.

In some cases, the step (b) solubilizes one or more organic acid inaddition to the monosaccharides and/or disaccharides.

In some cases, the one or more organic acid comprises at least one ofoxalate, tartrate, succinate, formate, citrate, malate, lactate oracetate.

In some cases, the total weight of oxalate, tartrate, succinate,formate, citrate, malate, lactate and acetate may be greater than 10% ofthe total weight of sucrose, maltose, lactose, glucose, fructose andgalactose in the portion solubilized and removed in step b.

In some cases, the physical pretreatment of step (a) comprises at leastone of chipping, chopping, milling, ball-milling, grinding, sprucing, orblending the biomass.

In some cases, the gentle pretreatment of step (b) occurs in an aqueoussolution may comprise water.

In some cases, the gentle pretreatment of step (b) occurs at atemperature of from 5° C. to 150° C.

In some cases, the gentle pretreatment of step (b) may be conducted from15 minutes to 1 hour.

In some cases, the strong pretreatment of step (c) comprises heating thegently pretreated biomass in the acidic solution or the alkali solution.

In some cases, the heating may be at a temperature of from 50° C. to150° C.

In some cases, the heating may be conducted from 30 minutes to 4 hours.

In some cases, the step (c) comprises treating the gently pretreatedbiomass in an alkali solution having a pH from 8 to 11.

In some cases, the alkali solution comprises at least one of sodiumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,aqueous ammonia, ammonium sulfate, or ammonium hydroxide.

In some cases, the step (c) comprises treating the gently pretreatedbiomass in an acidic solution having a pH from 4 to 6.

In some cases, the acidic solution comprises at least one of sulfuricacid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid,maleic acid, fumaric acid, or oxalic acid.

In some cases, the biomass comprises at least one of sugar cane, cornstover, corncob, wheat bran, wheat straw, hardwood, or softwood.

In some cases, the biomass comprises at least one of cellulose, chitin,chitosan, xylan, xyloglucan, mixed-linkage glucan, mannan, orlignocellulose.

In some cases, the one or more polysaccharide-cleaving enzymes comprisesat least one of cellulase, xylanase, xyloglucanase, endo-glucanase,cellobiohydrolase, mannanase, lichenase, or lytic polysaccharidemonooxygenase (LPMO).

In some cases, the one or more polysaccharide-cleaving enzymes comprisesat least one of AA9, AA10, AA11, AA13, AA14, or AA15.

In some cases, the one or more of the polysaccharide-cleaving enzymesmay be prepared from a filamentous fungi, such as Trichoderma reesei.

In some cases, the one or more polysaccharide-cleaving enzymes may beoperably linked to a catalytic module.

In some cases, the one or more polysaccharide-cleaving enzymes may beoperably linked to a non-catalytic module.

In some cases, the non-catalytic module may be a carbohydrate-bindingmodule.

In some cases, a water-soluble composition for human consumption maycomprise at least one of the following oligosaccharides: i) acello-oligosaccharide having a degree of polymerization (DP) of from twoto six; ii) a xylo-oligosaccharide having a DP of from two to twelve;iii) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; iv) a manno-oligosaccharide having a DP of from two to twelve;v) a mixed-linkage glucan oligosaccharide having a DP of from two tofive; vi) a xyloglucan oligosaccharide having a DP of from four totwelve; or vii) a chito-oligosaccharide having a DP of from two totwelve; and at least one of the following monosaccharides ordisaccharides: sucrose, maltose, lactose, fructose, or galactose,wherein the total dry weight of the monosaccharides or disaccharidescomprises less than 10% of the total dry weight of the oligosaccharideshaving a DP of from two to twelve.

In some cases, the ingredient may comprise at least two of theoligosaccharides listed in (i) to (vii). In some cases, the ingredientmay further comprise at least one of the following organic acids:oxalate, tartrate, succinate, formate, citrate, malate, lactate, oracetate. In some cases, the ingredient may comprise at least two of theorganic acids. In some cases, the ingredient may comprise at least twoof the oligosaccharides listed in (i) to (vii) in a ratio from 1:9 to1:1 in relation to each other.

In some cases, a composition may comprise at least one monosaccharide ordisaccharide selected from the group consisting of: glucose, fructose,or sucrose; at least one organic acid selected from the group consistingof oxalate, tartrate, succinate, formate, citrate, malate, lactate, oracetate, wherein the total weight of the organic acids is greater than10% of the total weight of the monosaccharides or disaccharides.

In some cases, a method for producing an ingredient for humanconsumption may comprise: (a) administering a physical pretreatment to abiomass to reduce an average size of the biomass; (b) administering agentle pretreatment to the physically pretreated biomass, the gentlepretreatment comprising: (i) incubating the physically pretreatedbiomass in a water solution to solubilize monosaccharides and/ordisaccharides from the physically pretreated biomass; and (ii) removinga portion of the solubilized monosaccharides and/or disaccharides fromthe water solution; (c) administering a strong pretreatment to thegently pretreated biomass to solubilize polysaccharides and to increasethe digestibility of the plant biomass; (d) isolating one or moresolubilized polysaccharides from the biomass; (e) contacting theremaining biomass with one or more enzymes to form one or moreoligosaccharides; (f) isolating the one or more oligosaccharides; and(g) combining a portion of the one or more soluble polysaccharides fromstep (d) with a portion of the one or more oligosaccharides from step(f) to form the ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “figure” and “FIG.” herein), of which:

FIG. 1 depicts the results of a thin-layer chromatography (TLC) analysisshowing the monosaccharide and disaccharide content of the indicatedbiomasses after one to five washing cycles (incubation pretreatments).

FIG. 2 depicts the results of a TLC analysis comparing the levels ofmonosaccharides and disaccharides in an enzyme hydrolysate productmixture when washing (incubation pretreatment) has (+) and has not (−)been performed.

FIG. 3 depicts the results of making a glaze with compositions of thepresent disclosure and a comparative composition.

FIG. 4 depicts baked products using a liquid ingredient of the presentdisclosure.

FIG. 5 depicts a high-performance anion exchange chromatography (HPAEC)analysis of Sample 4.

FIG. 6 depicts cereal bars produced using a liquid ingredient of thepresent disclosure.

FIG. 7 depicts an HPAEC chromatogram of the saccharides in the waterpost-washing of corncobs.

FIG. 8 is a simplified flow diagram depicting a method of treatingbiomass according to some embodiments of the present disclosure.

FIG. 9 is a simplified flow diagram depicting another method of treatingbiomass according to some embodiments of the present disclosure.

FIG. 10A is a diagram illustrating methods of processing biomass.

FIG. 10B depicts measurement of saccharides in multiple samples.

FIG. 10C depicts measurement of organic acids in multiple samples.

FIG. 10D depicts and visual observation of the samples processedaccording to the methods of FIG. 10A.

FIG. 11A illustrates a comparison between a cereal bar made usingsoluble polysaccharides and a cereal bar made using insolublepolysaccharides.

FIG. 11B depicts the hardness and stickiness of the cereal bars of FIG.11A using a penetration test.

FIG. 11C depicts the hardness of the cereal bars of FIG. 11A using acutting test.

DETAILED DESCRIPTION Introduction

Provided herein are methods of forming one or more ingredients. Themethods may include isolating one or more soluble polysaccharides from abiomass or feedstock (e.g., a plant biomass). The remaining biomass maythen be contacted or treated with one or more enzymes to form one ormore oligosaccharides and the one or more oligosaccharides may beenriched or isolated. Furthermore, at least a portion of the one or moresoluble polysaccharides isolated from the biomass may be combined with aportion of the one or more enriched or isolated oligosaccharides to formthe ingredient.

The texture of the ingredient may be consistent and smooth. Furthermore,the ingredient may have certain properties such that the ingredient maybe used as a sweetener and/or a sugar substitute. Properties of theingredient can include sweetness, smooth texture, desirable mouthfeel,ability to bind, ability to glaze or form a glaze, moistness, viscosity,ability to bulk, and/or ability to caramelize. In comparison to cornsyrup or high-fructose corn syrup, the ingredient can also have fewercalories, reduced glycemic index, reduced glycemic load, increasedfiber, and/or reduced sugar.

Also provided herein are methods for producing an ingredient forincorporation into a foodstuff, a nutraceutical, and/or a cosmetic,wherein the methods may include one or more pretreatment steps performedon a biomass. For example, the method may include a first pretreatmentstep, a second pretreatment step, a third pretreatment step, oradditional pretreatment steps performed on a biomass. The pretreatmentsteps may be performed in a specified order.

The biomass used to produce one or more oligosaccharides may be a plantbiomass. Examples of plant biomass include, but are not limited to,sugar cane, corn stover, corncob, wheat bran, wheat straw, hardwood, orsoftwood. In some cases, the biomass may comprise cellulose, chitin,chitosan, xylan, xyloglucan, mixed-linkage glucan, mannan, orlignocellulose.

The biomass may be digested into one or more oligosaccharides. Thebiomass digestion may be performed enzymatically. The enzymaticdigestion may be performed after one or more pretreatment steps. The oneor more pretreatment steps may be performed to reduce the size of abiomass and/or increase the surface area of the biomass available fordigestion. The one or more pretreatment steps may include one or morewashing steps, solubilizing steps, or pre-digestion treatments. In somecases, one or more pretreatment steps may be performed to reduce themonosaccharides and/or disaccharides present in the biomass. In somecases, one or more pretreatment steps may be performed to recover asoluble polysaccharide fraction from the biomass.

During the one or more pretreatment steps, monosaccharides and/ordisaccharides may be removed from a starting material (e.g., a biomass).Stated another way, monosaccharides and/or disaccharides may be removedfrom the biomass during one or more of the pretreatment steps.Accordingly, no, or substantially no, monosaccharides and/ordisaccharides may be yielded upon completion of the one or morepretreatment steps. That is, the pretreated biomass may comprise no orsubstantially no monosaccharides and/or disaccharides. This can improvethe efficiency of the method for producing the ingredient forincorporation into a foodstuff, a nutraceutical, and/or a cosmetic asprovided herein. For example, as a portion of the monosaccharides and/ordisaccharides has already been removed from the biomass during the oneor more pretreatment steps, less purification of the ingredient (e.g.,to remove monosaccharides and/or disaccharides) may be needed.Specifically, fewer filtration steps, or less stringent filtrationsteps, may be needed during purification to generate the ingredient asdisclosed herein.

A first pretreatment step (pretreatment step 1) may include physicallytreating a biomass (e.g., chipping the biomass). A second pretreatmentstep (pretreatment step 2 or gentle pretreatment) may include subjectingthe physically treated biomass to an incubation cycle or a washingcycle. The incubation cycle may include incubating the physicallytreated biomass (from pretreatment step 1) in an aqueous solution tosolubilize monosaccharides and/or disaccharides from the physicallytreated biomass. The incubation cycle may also include removing aportion of the solubilized monosaccharides and/or disaccharides from theaqueous solution. In some cases, the aqueous solution may include water.In some other cases, the incubation cycle may be performed at about 25°C. for a period of about 30 minutes to about 1.5 hours. Pretreatmentstep 2 may be a gentle pretreatment step. For example, the conditions(e.g., solution, temperature, time, etc.) of pretreatment step 2 may begentler than the conditions of pretreatment step 3 as described infurther detail below.

A third pretreatment step (pretreatment step 3 or strong pretreatment)may include treating the incubated biomass from pretreatment step 2 inone of an acidic solution or an alkali solution. Pretreatment step 3 canimprove the digestibility of the biomass (e.g., by an enzyme).Pretreatment step 3 may also improve enzyme access to the biomass. Invarious instances, pretreatment step 3 may occur in an alkali solution(e.g., a 1% w/v NaOH solution) at a temperature above room temperature(e.g., at from about 90° C. to about 110° C.). Furthermore, pretreatmentstep 3 may be conducted by being held at the desired temperature forabout 30 minutes to 1 hour. For instance, the effective temperature, inthis example 90° C., is held for an hour (this can be altered dependingon the desired characteristic). The solution in the third pretreatmentstep can be retained. Stated another way, the solution may not bediscarded as the treated biomass moves from the pretreatment steps tothe steps after the pretreatment steps as described in further detailbelow. In some cases, pretreatment step 3 may include a thermochemicaltreatment. That is, pretreatment step 3 may be performed in an acidic oralkali solution and/or pretreatment step 3 may be performed at atemperature above room temperature.

In various instances, after the one or more pretreatment steps, themethod for producing an ingredient for human consumption may includecontacting, in a solution or suspension, one or morepolysaccharide-cleaving enzymes and the biomass from pretreatment step 3to form one or more oligosaccharides. The method may further includeenriching the solution or suspension to increase the concentration ofthe one or more oligosaccharides to form the ingredient (e.g., theingredient for human consumption).

The pretreatment steps may increase the efficiency of the method incomparison to some other methods. The efficiency may be improved insofaras less extensive downstream processing may be needed. In some cases,downstream processing may include the process of removingmonosaccharides and/or disaccharides from the pretreated biomass. Insome embodiments, it may be difficult to remove disaccharides alone, forexample, from an intermediate solution or fraction that is generated bythe enzyme digestion. Thus, it may be more efficient to remove thedisaccharides during the pretreatment steps.

The steps of the downstream processing can include ion-exchangechromatography, ultrafiltration, microfiltration, nanofiltration, etc.Part of the role of the nanofiltration step may be to remove excessmonosaccharides from the oligosaccharide mixture. This nanofiltrationmay be performed multiple times to arrive at desirable monosaccharidelevels. The number of such nanofiltration steps may be reduced whenthere are fewer monosaccharides in the pretreated biomass (e.g., whenthe monosaccharides have been removed by washing or incubation).Furthermore, ultrafiltration generally cannot differentiate betweendesirable disaccharides (e.g., cellobiose) and undesirabledisaccharides. Accordingly, removing the undesirable disaccharidesduring pretreatment and generating the desirable disaccharides duringthe enzyme treatment step can also reduce the number of steps involvedor needed during downstream processing.

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a sample” includes a plurality ofsamples, including mixtures thereof.

The term “about,” as used herein, can mean within 1 or more than 1standard deviation. Alternatively, about can mean a range of up to 10%,up to 5%, or up to 1% of a given value. For example, about can mean upto ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a givenvalue.

As used herein, “food” and “foodstuff” generally refer to any itemdestined for consumption, which may be consumption by a human or by anyother animal. It may be food, feed, beverage, or an ingredient to beused in the production of any of the above.

As used herein, “nutraceutical” generally refers to any compositionintroduced into a human or other animal, whether by ingestion,injection, absorption, or any other method, for the purpose of providingnutrition to the human or other animal. Use of such a nutraceutical maytake the form of a drink with added dietary fiber, a prebiotic additive,a pill or other capsule, or any other suitable use.

As used herein, “cosmetic” generally refers to any composition which isintended for use on humans or other animals to increase their aestheticappeal or prevent future loss of aesthetic appeal, as well as any othercompositions known in general parlance as cosmetics. Aesthetic appeal isnot limited to visual aesthetics but applies as well to textural or anyother appeal. The cosmetic may be mascara, foundation, lip gloss,eyeshadow, eyeliner, primer, lipstick, blush, nail polish, bronzer, orany other makeup; shampoo, conditioner, styling mousse, styling gel,hairspray, hair dye, hair wax, or any other hair product; moisturizer,exfoliant, sun cream, cleanser, toothpaste, cream, lotion, ointment, orany other composition effective in modifying teeth, skin, hair, or otherparts of the body in some aesthetic way. Further, the cosmetic may be acomposition used as a component of a face mask, brush, hair roller,other styling device, other solid structure, or any other suitablecomposition.

As used herein, “ingredient” generally refers to any compositionsuitable for incorporation into a foodstuff, cosmetic, or nutraceuticalproduct, which may include those which are used directly as the productitself. It may be a dry or liquid ingredient, unless it is specificallyreferred to as “dry” or “liquid.” This includes compositions that may bedeemed to be an intermediate during a method of the disclosure, such asa composition formed after the combining of the one or moreoligosaccharides and the one or more soluble polysaccharides prior toany further purification, optimization, drying, dissolving, or any othersuch steps, as well as including the final composition obtained from themethod.

As used herein, “polysaccharide” generally refers to a saccharidepolymer of any length greater than about 20 residues. Polysaccharidesmay be highly branched, lightly branched, or unbranched. Polysaccharidesmay include any manner of glycosidic bond in any combination; any numberof, for example, α or β linkages; and any combination of monomer types,such as glucose, glucosamine, mannose, xylose, galactose, fucose,fructose, glucuronic acid, arabinose, or derivatives thereof, such asany combination of the above monomers decorated with acetyl or othergroups. The polysaccharide may be a cellulosic or hemicellulosicpolymer. Hemicellulosic polymers envisaged include xylan,glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. In someembodiments, the cellulosic polymer may be cellulose.

As used herein, “lignocellulose” generally refers topolysaccharide-comprising aggregates that are, or are derived from,plant cell wall material. For example, they may include one or more ofthe following polysaccharides associated together: cellulose, xylan,mannan, and mixed-linkage glucan.

As used herein “highly branched,” “lightly branched,” and “unbranched”generally refer to the number of side-chains per stretch of main chainin a saccharide. Highly branched saccharides have on average from 4 to10 side chains per 10 main-chain residues, slightly branched saccharideshave on average from 1 to 3 side chains per 10 main-chain residues, andunbranched saccharides have only one main chain and no side chains. Theaverage is calculated by dividing the number of side chains in asaccharide by the number of main-chain residues.

As used herein, “saccharide” generally refers to any polysaccharideand/or oligosaccharide, such as a monosaccharide and/or a disaccharide.

As used herein, “oligosaccharide” generally refers to saccharidepolymers having chain lengths less than or equal to about 20 saccharideresidues. Oligosaccharides may be highly branched, lightly branched, orunbranched; and may include glycosidic bonds in any combination, anynumber of α or β linkages, and any combination of monomer types, such asglucose, glucosamine, mannose, xylose, galactose, fucose, fructose,glucuronic acid, arabinose, or derivatives thereof. Suitable derivativesinclude the above monomers including acetyl or other groups.

As used herein, “monosaccharide” and “disaccharide” generally refer tosaccharide compounds consisting of one or two residues, respectively.Monosaccharides are compounds such as glucose, glucosamine, xylose,galactose, fucose, fructose, glucuronic acid, arabinose, galacturonicacid, or epimers or other derivatives thereof. Suitable derivativesinclude acetyl or other groups. Disaccharides are compounds consistingof two monosaccharides joined via any glycosidic bond.

As used herein, “cello-oligosaccharides” generally refer tooligosaccharides composed of one or more glucose residues linked byβ-1,4-glycosidic bonds, and may be chemically related to that byoxidation, reduction, esterification, epimerization, or another chemicalmodification.

As used herein, “xylo-oligosaccharides” generally refer tooligosaccharides composed primarily of xylose residues (typically linkedby β-1,4-glycosidic bonds) and may also contain glucuronic acid residuesand/or arabinose residues and/or acetyl groups and/or any othermodification, and may be chemically related to that by oxidation,reduction, esterification, epimerization, further glycosylation, oranother chemical modification.

As used herein, “arabinoxylo-oligosaccharides” generally refer tooligosaccharides composed of xylose residues (typically linked byβ-(1→4)-bonds substituted with arabinose side-chains), typically linkedby (1→2)-bonds or (1→3)-bonds) and may be chemically related to that byoxidation, reduction, esterification, epimerization, furtherglycosylation, or another chemical modification.

As used herein, “mixed-linkage glucan-oligosaccharides” generally referto oligosaccharides composed of one or more glucose residues linked byat least one β-1,3-glycosidic bond and at least one β-1,4-glycosidicbond, and may be chemically related to that by oxidation, reduction,esterification, epimerization, or another chemical modification

As used herein, “manno-oligosaccharides” generally refer tooligosaccharides composed of one or more mannose residues and optionallycontaining one or more glucose and/or galactose residues, and may bechemically related to that by oxidation, reduction, esterification,epimerization, or another chemical modification.

As used herein, “chito-oligosaccharides” generally refer tooligosaccharides composed of one or more glucosamine and/orN-acetyl-glucosamine residues, and may be chemically related to that byoxidation, reduction, esterification, epimerization, or another chemicalmodification.

As used herein, “cellulose” generally refers to polysaccharides composedof glucose residues linked by β-1,4-glycosidic bonds, and derivativesthereof. As used herein, “xylan” generally refers to polysaccharidescomposed of a backbone of xylose residues and may also containglucuronic acid residues and/or arabinose residues and/or acetyl groupsand/or any other modification. As used herein, “mixed-linkage glucan”generally refers to polysaccharides composed of glucose residues linkedby β-1,3-glycosidic bonds and β-1,4-glycosidic bonds. As used herein,“mannan” generally refers to polysaccharides composed of greater than40% mannose residues and optionally containing glucose and/or galactoseresidues. As used herein, “chitin” or “chitosan” generally refer topolysaccharides composed of glucosamine and/or N-acetyl-glucosamineresidues. The polysaccharides of cellulose, xylan, mixed-linkage glucan,mannan, chitin, or chitosan may include chemical variants that have beenmodified by oxidation, reduction, esterification, epimerization, oranother chemical modification.

As used herein, “soluble,” “solubility,” and grammatical variantsthereof generally refer to solubility in an aqueous solution (e.g.,water). As used herein, “solubilize” generally refers to a solidbecoming incorporated into an aqueous solution or a liquid so as to forma solution.

As used herein, “suspension” generally refers to a compositioncomprising at least two immiscible phases, for example, a solid phaseand a liquid phase, wherein the weight of the solid phase may be, as apercentage of the weight of the composition, in the range of from about0.5% to about 30%, about 1% to about 20%, about 2% to about 15%, orabout 3% to about 10%. The suspension may comprise a suitable solvent,which may be water.

As used herein, “viscosity” generally refers to a quantity expressingthe magnitude of internal friction in a fluid, as measured by the forceper unit area resisting uniform flow. The viscosity can be measured by avariety of methods, but the values given herein, unless indicatedotherwise, refer to those obtained by using a Brookfield HDB VEroto-viscometer using standard testing procedures, operated as per themanufacturer's instructions with respect to ranges, and with a 400 mLsample taken in a tall-form beaker to ensure that no container effectsoccur.

As used herein, “dissolved” generally refers to a solid becomingincorporated into a liquid so as to form a solution.

I. Pretreatment Physical Pretreatment

A mechanical or physical pretreatment may be performed on a biomass forthe digestion of the biomass into one or more oligosaccharides. Themechanical and/or physical pretreatment may be the first pretreatmentstep in the process of biomass digestion. Alternatively, the mechanicalor physical pretreatment step may be performed after anotherpretreatment step. For instance, a mechanical or physical pretreatmentstep may be performed after another pretreatment step, such as a washingpretreatment step.

A biomass may be mechanically or physically pretreated to reduce thesize of the biomass. Examples of mechanical or physical pretreatmentsteps include, but are not limited to: chipping, chopping, milling,ball-milling, grinding, sprucing, blending, and/or steam explosion ofthe biomass. More than one physical pretreatment may be performed on thebiomass.

Solubilizing Step

The biomass may undergo a solubilizing step. The solubilizing step maybe, or be a portion of, a gentle pretreatment step, which solubilizes apolysaccharide fraction or a monosaccharide and/or disaccharide fractionfrom the biomass. The solubilizing pretreatment step may be a washingstep, an incubation step, a thermochemical step, or a chemical treatmentstep. The solubilizing pretreatment may be performed before a physicalor mechanical pretreatment step. The solubilizing pretreatment may beperformed after a physical or mechanical pretreatment step.

In some embodiments, the solubilizing step may be performed to remove afraction of soluble polysaccharides. Soluble polysaccharides can beadded to one or more oligosaccharides, for example, upon purification ofthe soluble polysaccharides. The soluble polysaccharide fraction may beused to produce food types of desired taste, texture, quality,adhesiveness, and smell. Presence of solubilized polysaccharides mayalso help produce a better-quality product which does not producesediments (or graininess) in a food product.

The solubilizing step may be a chemical or thermochemical treatment ofthe biomass. The chemical or thermochemical treatment may comprise oneor more aqueous solutions. The aqueous solution may comprise one or moresalts, acids, alkalis, or ions. The aqueous solution may comprise one ormore of sodium hydroxide, potassium hydroxide, sodium carbonate, calciumcarbonate, calcium hydroxide, ammonium sulfate, ammonium hydroxide,aqueous ammonia, dilute sulfuric acid, dilute acetic acid, dilutehydrochloric acid, or dilute phosphoric acid.

The aqueous solution may be an alkali solution with a pH from 10 to 14.The aqueous solution may be an alkali solution with a pH from 10 to 11,10 to 12, 10 to 13, 10 to 14, 11 to 12, 11 to 13, 11 to 14, 12 to 13, 12to 14, or 13 to 14. The aqueous solution may be an acid solution with apH from 2 to 6. The aqueous solution may be an acid solution with a pHfrom 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4to 6, or 5 to 6.

The solubilization step may be performed at a temperature from 30° C. to180° C. The solubilization step may be performed at a temperature of atleast 30° C. The solubilization step may be performed at a temperatureof at most 180° C. The solubilization step may be performed at atemperature from 30° C. to 60° C., 30° C. to 90° C., 30° C. to 120° C.,30° C. to 150° C., 30° C. to 180° C., 60° C. to 90° C., 60° C. to 120°C., 60° C. to 150° C., 60° C. to 180° C., 90° C. to 120° C., 90° C. to150° C., 90° C. to 180° C., 120° C. to 150° C., 120° C. to 180° C., or150° C. to 180° C. The solubilization step may be performed at atemperature of at least 30° C., 60° C., 90° C., 120° C., 150° C., or180° C.

The duration of the solubilizing step may be altered according to thebiomass being used, the soluble polysaccharide component desired, and/orthe complexity of the soluble polysaccharide desired. The solubilizingstep may be performed from about 10 minutes to about 24 hours. Thesolubilization step may be performed for at least 10 minutes. Thesolubilization step may be performed for at most 60 minutes. Thesolubilization step may be performed from 10 minutes to 30 minutes, 10minutes to 60 minutes, or 30 minutes to 60 minutes. The solubilizationstep may be performed for at least 10 minutes, 30 minutes, or 60minutes. The solubilization step may be performed for 1 hour to 24hours. The solubilization step may be performed for at least 1 hour. Thesolubilization step may be performed for at most 24 hours. Thesolubilization step may be performed for 1 hour to 4 hours, 1 hour to 8hours, 1 hour to 12 hours, 1 hour to 16 hours, 1 hour to 20 hours, 1hour to 24 hours, 4 hours to 8 hours, 4 hours to 12 hours, 4 hours to 16hours, 4 hours to 20 hours, 4 hours to 24 hours, 8 hours to 12 hours, 8hours to 16 hours, 8 hours to 20 hours, 8 hours to 24 hours, 12 hours to16 hours, 12 hours to 20 hours, 12 hours to 24 hours, 16 hours to 20hours, 16 hours to 24 hours, or 20 hours to 24 hours. The solubilizationstep may be performed for at least 1 hour, 4 hours, 8 hours, 12 hours,16 hours, 20 hours, or 24 hours.

Soluble polysaccharides may be removed from the solution after asolubilizing step. A fraction of the solubilized polysaccharides may beremoved from the solubilized fraction. In some cases, at least 5% of thesolubilized polysaccharides may be removed and/or purified. In somecases, at most 100% of the solubilized polysaccharides may be removedand/or purified. In some cases, 5% to 10%, 5% to 20%, 5% to 30%, 5% to40%, 5% to 60%, 5% to 80%, 5% to 90%, 5% to 100%, 10% to 20%, 10% to30%, 10% to 40%, 10% to 60%, 10% to 80%, 10% to 90%, 10% to 100%, 20% to30%, 20% to 40%, 20% to 60%, 20% to 80%, 20% to 100%, 30% to 40%, 30% to60%, 30% to 80%, 30% to 100%, 40% to 60%, 40% to 80%, 40% to 100%, 60%to 80%, or 60% to 100% of the solubilized polysaccharides may be removedand purified. In some cases, at least about 5%, 10%, 20%, 30%, 40%, 60%,80%, or 100% of the solubilized polysaccharides may be removed andpurified.

Gentle Pretreatment

The biomass may undergo a gentle pretreatment step. The gentlepretreatment step may solubilize a polysaccharide fraction or amonosaccharide and/or disaccharide fraction from the biomass. In somecases, the gentle pretreatment step may include at least a portion, orall, of the solubilizing step. Stated another way, the solubilizing stepmay be a portion or sub-step of the gentle pretreatment step. The gentlepretreatment step may be a washing step, an incubation step, athermochemical step, or a chemical treatment step. The gentlepretreatment may be performed before a physical or mechanicalpretreatment step. The gentle pretreatment may be performed after aphysical or mechanical pretreatment step. The gentle pretreatment may beperformed simultaneously with a physical or mechanical pretreatmentstep. The gentle pretreatment step may be performed one or more times.The gentle pretreatment may be performed 2, 3, 4, 5, 6, 7, 8, 9, or 10times.

The gentle pretreatment step may be an incubation step or a washingstep. The biomass (physically treated or untreated) may be incubated inan aqueous solution. The aqueous solution may be water, or it maycomprise salts, acids, alkali, ions, alcohols, and/or other chemicals.The pH of the aqueous solution may be from 6.2 to 8.5. The pH of theaqueous solution may be at least 6.2. The pH of the aqueous solution maybe at most 8.5. The pH of the aqueous solution may be from 6.2 to 6.5,6.2 to 7, 6.2 to 7.2, 6.2 to 7.5, 6.2 to 7.7, 6.2 to 8, 6.2 to 8.2, 6.2to 8.5, 6.5 to 7, 6.5 to 7.2, 6.5 to 7.5, 6.5 to 7.7, 6.5 to 8, 6.5 to8.2, 6.5 to 8.5, 7 to 7.2, 7 to 7.5, 7 to 7.7, 7 to 8, 7 to 8.2, 7 to8.5, 7.2 to 7.5, 7.2 to 7.7, 7.2 to 8, 7.2 to 8.2, 7.2 to 8.5, 7.5 to7.7, 7.5 to 8, 7.5 to 8.2, 7.5 to 8.5, 7.7 to 8, 7.7 to 8.2, 7.7 to 8.5,8 to 8.2, 8 to 8.5, or 8.2 to 8.5. The pH of the aqueous solution may befrom 6.2, 6.5, 7, 7.2, 7.5, 7.7, 8, 8.2, or 8.5.

The pH of the aqueous solution may be from 9 to 12. The pH of theaqueous solution may be from at least 9. The pH of the aqueous solutionmay be from at most 12. The pH of the aqueous solution may be from 9 to9.5, 9 to 10, 9 to 10.5, 9 to 11, 9 to 11.5, 9 to 12, 9.5 to 10, 9.5 to10.5, 9.5 to 11, 9.5 to 11.5, 9.5 to 12, 10 to 10.5, 10 to 11, 10 to11.5, 10 to 12, 10.5 to 11, 10.5 to 11.5, 10.5 to 12, 11 to 11.5, 11 to12, or 11.5 to 12. The pH of the aqueous solution may be from 9, 9.5,10, 10.5, 11, 11.5, or 12.

The incubation step may be performed for 15 minutes to 60 minutes. Theincubation step may be performed for at least 15 minutes. The incubationstep may be performed for at most 60 minutes. The incubation step may beperformed for 15 minutes to 30 minutes, 15 minutes to 45 minutes, 15minutes to 60 minutes, 30 minutes to 45 minutes, 30 minutes to 60minutes, or 45 minutes to 60 minutes. The incubation step may beperformed for at least 15 minutes, 30 minutes, 45 minutes, or 60minutes. The incubation step may be performed for 1 hour to 24 hours.The incubation step may be performed for at least 1 hour. The incubationstep may be performed for at most 24 hours. The incubation step may beperformed for 1 hour to 4 hours, 1 hour to 8 hours, 1 hour to 12 hours,1 hour to 16 hours, 1 hour to 20 hours, 1 hour to 24 hours, 4 hours to 8hours, 4 hours to 12 hours, 4 hours to 16 hours, 4 hours to 20 hours, 4hours to 24 hours, 8 hours to 12 hours, 8 hours to 16 hours, 8 hours to20 hours, 8 hours to 24 hours, 12 hours to 16 hours, 12 hours to 20hours, 12 hours to 24 hours, 16 hours to 20 hours, 16 hours to 24 hours,or 20 hours to 24 hours. The incubation step may be performed for atleast 1 hour, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24hours.

Solubilized polysaccharides, monosaccharides, and/or disaccharides maybe removed from the aqueous solution after the gentle pretreatment step.In some cases, monosaccharides and/or disaccharides may be removed fromthe aqueous solution after the gentle pretreatment step.

Soluble polysaccharides may be removed from the solution after a gentlepretreatment step. At least a portion of the solubilized polysaccharidesmay be removed from the solubilized fraction. In some cases, at least 5%of the solubilized polysaccharides may be removed and/or purified. Insome cases, at most 100% of the solubilized polysaccharides may beremoved and/or purified. In some cases, 5% to 10%, 5% to 20%, 5% to 30%,5% to 40%, 5% to 60%, 5% to 80%, 5% to 100%, 10% to 20%, 10% to 30%, 10%to 40%, 10% to 60%, 10% to 80%, 10% to 100%, 20% to 30%, 20% to 40%, 20%to 60%, 20% to 80%, 20% to 100%, 30% to 40%, 30% to 60%, 30% to 80%, 30%to 100%, 40% to 60%, 40% to 80%, 40% to 100%, 60% to 80%, or 60% to 100%of the solubilized polysaccharides may be removed and/or purified. Insome cases, at least about 5%, 10%, 20%, 30%, 40%, 60%, 80%, or 100% ofthe solubilized polysaccharides may be removed and/or purified after thegentle pretreatment step.

Monosaccharides and/or disaccharides may be removed from the solutionafter a gentle pretreatment step. A fraction of the solubilizedmonosaccharides and/or disaccharides may be removed from the aqueoussolution after the incubation step. In some cases, at least 5% of thesolubilized monosaccharides and/or disaccharides may be removed. In somecases, at most 100% of the solubilized monosaccharides and/ordisaccharides may be removed. In some cases, 5% to 10%, 5% to 20%, 5% to30%, 5% to 40%, 5% to 60%, 5% to 80%, 5% to 100%, 10% to 20%, 10% to30%, 10% to 40%, 10% to 60%, 10% to 80%, 10% to 100%, 20% to 30%, 20% to40%, 20% to 60%, 20% to 80%, 20% to 100%, 30% to 40%, 30% to 60%, 30% to80%, 30% to 100%, 40% to 60%, 40% to 80%, 40% to 100%, or 60% to 100% ofthe solubilized monosaccharides and/or disaccharides may be removed. Insome cases, at least about 5%, 10%, 20%, 30%, 40%, 60%, 80%, or 100% ofthe solubilized monosaccharides and/or disaccharides may be removedand/or purified after the gentle pretreatment step. The monosaccharideand/or disaccharide portion may be discarded after the incubation step.In certain instances, the portion of the solubilized monosaccharidesand/or disaccharides removed in this step may not be combined with theportion of the one or more oligosaccharides produced in the biomasstreatment.

The aqueous solution may be removed from the biomass after the gentlepretreatment step. A portion of the aqueous solution may be removed fromthe biomass after the gentle pretreatment step. 5% to 100% of theaqueous solution may be removed from the biomass after the gentlepretreatment step. At least 5% of the aqueous solution may be removedfrom the biomass after the gentle pretreatment step. At most 98% of theaqueous solution may be removed from the biomass after the gentlepretreatment step. Five percent to 10%, 5% to 20%, 5% to 40%, 5% to 50%,5% to 60%, 5% to 80%, 5% to 90%, 5% to 98%, 10% to 20%, 10% to 40%, 10%to 50%, 10% to 60%, 10% to 80%, 10% to 90%, 10% to 98%, 20% to 40%, 20%to 50%, 20% to 60%, 20% to 80%, 20% to 90%, 20% to 98%, 40% to 50%, 40%to 60%, 40% to 80%, 40% to 90%, 40% to 98%, 50% to 60%, 50% to 80%, 50%to 90%, 50% to 98%, 60% to 80%, 60% to 90%, 60% to 98%, 80% to 90%, 80%to 98%, or 90% to 98% of the aqueous solution may be removed from thebiomass after the gentle pretreatment step. At least 5%, 10%, 20%, 40%,50%, 60%, 80%, 90%, or 98% of the aqueous solution may be removed fromthe biomass after the gentle pretreatment step. A portion of the aqueoussolution may be removed using a filter press, centrifugation,sedimentation, filtration, and/or any other suitable method.

Strong Pretreatment

The biomass may undergo a strong pretreatment step. The strongpretreatment step may solubilize a polysaccharide fraction or amonosaccharide and/or disaccharide fraction from the biomass. The strongpretreatment step may be performed to make the biomass more digestibleby enzymes. The strong pretreatment may help disrupt the hydrogen bondsin the biomass. The strong pretreatment step may be a washing step, athermochemical step, or a chemical treatment step. The strongpretreatment may be performed before a physical or mechanicalpretreatment step. The strong pretreatment step may be performed after aphysical or mechanical pretreatment step. The strong pretreatment stepmay be performed before a gentle pretreatment step. The strongpretreatment step may be performed after a gentle pretreatment step. Thestrong pretreatment step may be performed one or more times. The strongpretreatment may be performed 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

The strong pretreatment step may be a thermochemical treatment. Thechemical or thermochemical treatment may comprise one or more aqueoussolutions. The aqueous solution may comprise one or more salts, acids,alkalis, or ions. The aqueous solution may be an alkali solutioncomprising one or more of sodium hydroxide, potassium hydroxide, sodiumcarbonate, calcium carbonate, calcium hydroxide, ammonium sulfate,ammonium hydroxide, or aqueous ammonia. The aqueous solution may be anacidic solution comprising at least one of sulfuric acid, hydrochloricacid, nitric acid, phosphoric acid, acetic acid, maleic acid, fumaricacid, or oxalic acid. In some embodiments, upon completion of the one ormore strong pretreatment steps, the aqueous solution may be retained. Inother words, the aqueous solution may not be discarded or rejected.

The thermochemical pretreatment may be performed at a pH of from about 2to 6.5. The thermochemical pretreatment may be performed at a pH of atleast 2. The thermochemical pretreatment may be performed at a pH of atmost 6.5. The thermochemical pretreatment may be performed at a pH of 2to 2.5, 2 to 3, 2 to 3.5, 2 to 4, 2 to 4.5, 2 to 5, 2 to 5.5, 2 to 6, 2to 6.5, 2.5 to 3, 2.5 to 3.5, 2.5 to 4, 2.5 to 4.5, 2.5 to 5, 2.5 to5.5, 2.5 to 6, 2.5 to 6.5, 3 to 3.5, 3 to 4, 3 to 4.5, 3 to 5, 3 to 5.5,3 to 6, 3 to 6.5, 3.5 to 4, 3.5 to 4.5, 3.5 to 5, 3.5 to 5.5, 3.5 to 6,3.5 to 6.5, 4 to 4.5, 4 to 5, 4 to 5.5, 4 to 6, 4 to 6.5, 4.5 to 5, 4.5to 5.5, 4.5 to 6, 4.5 to 6.5, 5 to 5.5, 5 to 6, 5 to 6.5, 5.5 to 6, 5.5to 6.5, or 6 to 6.5. The thermochemical pretreatment may be performed ata pH of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5.

The thermochemical pretreatment may be performed at a pH of 7.5 to 12.The thermochemical pretreatment may be performed at a pH of at least7.5. The thermochemical pretreatment may be performed at a pH of at most12. The thermochemical pretreatment may be performed at a pH of 7.5 to8, 7.5 to 8.5, 7.5 to 9, 7.5 to 9.5, 7.5 to 10, 7.5 to 10.5, 7.5 to 11,7.5 to 11.5, 7.5 to 12, 8 to 8.5, 8 to 9, 8 to 9.5, 8 to 10, 8 to 10.5,8 to 11, 8 to 11.5, 8 to 12, 8.5 to 9, 8.5 to 9.5, 8.5 to 10, 8.5 to10.5, 8.5 to 11, 8.5 to 11.5, 8.5 to 12, 9 to 9.5, 9 to 10, 9 to 10.5, 9to 11, 9 to 11.5, 9 to 12, 9.5 to 10, 9.5 to 10.5, 9.5 to 11, 9.5 to11.5, 9.5 to 12, 10 to 10.5, 10 to 11, 10 to 11.5, 10 to 12, 10.5 to 11,10.5 to 11.5, 10.5 to 12, 11 to 11.5, 11 to 12, or 11.5 to 12. Thethermochemical pretreatment may be performed at a pH of about 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12.

The thermochemical pretreatment may be performed at a temperature of 50°C. to 150° C. The thermochemical pretreatment may be performed at atemperature of at least 50° C. The thermochemical pretreatment may beperformed at a temperature of at most 150° C. The thermochemicalpretreatment may be performed at a temperature of 50° C. to 75° C., 50°C. to 80° C., 50° C. to 90° C., 50° C. to 100° C., 50° C. to 120° C.,50° C. to 130° C., 50° C. to 150° C., 75° C. to 80° C., 75° C. to 90°C., 75° C. to 100° C., 75° C. to 120° C., 75° C. to 130° C., 75° C. to150° C., 80° C. to 90° C., 80° C. to 100° C., 80° C. to 120° C., 80° C.to 130° C., 80° C. to 150° C., 90° C. to 100° C., 90° C. to 120° C., 90°C. to 130° C., 90° C. to 150° C., 100° C. to 120° C., 100° C. to 130°C., 100° C. to 150° C., 120° C. to 130° C., 120° C. to 150° C., or 130°C. to 150° C. The thermochemical pretreatment may be performed at atemperature of at least 50° C., 75° C., 80° C., 90° C., 100° C., 120°C., 130° C., or 150° C.

The thermochemical treatment may be performed for 0.5 hours to 4 hours.The thermochemical treatment may be performed for at least 0.5 hours.The thermochemical treatment may be performed for at most 4 hours. Thethermochemical treatment may be performed for 0.5 hours to 0.75 hours,0.5 hours to 1 hour, 0.5 hours to 1.5 hours, 0.5 hours to 2 hours, 0.5hours to 2.5 hours, 0.5 hours to 3 hours, 0.5 hours to 3.5 hours, 0.5hours to 4 hours, 0.75 hours to 1 hour, 0.75 hours to 1.5 hours, 0.75hours to 2 hours, 0.75 hours to 2.5 hours, 0.75 hours to 3 hours, 0.75hours to 3.5 hours, 0.75 hours to 4 hours, 1 hour to 1.5 hours, 1 hourto 2 hours, 1 hour to 2.5 hours, 1 hour to 3 hours, 1 hour to 3.5 hours,1 hour to 4 hours, 1.5 hours to 2 hours, 1.5 hours to 2.5 hours, 1.5hours to 3 hours, 1.5 hours to 3.5 hours, 1.5 hours to 4 hours, 2 hoursto 2.5 hours, 2 hours to 3 hours, 2 hours to 3.5 hours, 2 hours to 4hours, 2.5 hours to 3 hours, 2.5 hours to 3.5 hours, 2.5 hours to 4hours, 3 hours to 3.5 hours, 3 hours to 4 hours, or 3.5 hours to 4hours. The thermochemical treatment may be performed for at least 0.5hours, 0.75 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5hours, or 4 hours.

The thermochemically treated biomass may comprise less than 1% w/w to30% w/w monosaccharides. The thermochemically treated biomass maycomprise less than 1% w/w to 2% w/w, 1% w/w to 5% w/w, 1% w/w to 10%w/w, 1% w/w to 15% w/w, 1% w/w to 20% w/w, 1% w/w to 25% w/w, 1% w/w to30% w/w, 2% w/w to 5% w/w, 2% w/w to 10% w/w, 2% w/w to 15% w/w, 2% w/wto 20% w/w, 2% w/w to 25% w/w, 2% w/w to 30% w/w, 5% w/w to 10% w/w, 5%w/w to 15% w/w, 5% w/w to 20% w/w, 5% w/w to 25% w/w, 5% w/w to 30% w/w,10% w/w to 15% w/w, 10% w/w to 20% w/w, 10% w/w to 25% w/w, 10% w/w to30% w/w, 15% w/w to 20% w/w, 15% w/w to 25% w/w, 15% w/w to 30% w/w, 20%w/w to 25% w/w, 20% w/w to 30% w/w, or 25% w/w to 30% w/wmonosaccharides. The thermochemically treated biomass may comprise lessthan 1% w/w, 2% w/w, 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, or 30%w/w monosaccharides.

The thermochemically treated biomass may comprise 5% w/w to 50% w/wdisaccharides. The thermochemically treated biomass may comprise 5% w/wto 10% w/w, 5% w/w to 15% w/w, 5% w/w to 20% w/w, 5% w/w to 25% w/w, 5%w/w to 30% w/w, 5% w/w to 35% w/w, 5% w/w to 40% w/w, 5% w/w to 50% w/w,10% w/w to 15% w/w, 10% w/w to 20% w/w, 10% w/w to 25% w/w, 10% w/w to30% w/w, 10% w/w to 35% w/w, 10% w/w to 40% w/w, 10% w/w to 50% w/w, 15%w/w to 20% w/w, 15% w/w to 25% w/w, 15% w/w to 30% w/w, 15% w/w to 35%w/w, 15% w/w to 40% w/w, 15% w/w to 50% w/w, 20% w/w to 25% w/w, 20% w/wto 30% w/w, 20% w/w to 35% w/w, 20% w/w to 40% w/w, 20% w/w to 50% w/w,25% w/w to 30% w/w, 25% w/w to 35% w/w, 25% w/w to 40% w/w, 25% w/w to50% w/w, 30% w/w to 35% w/w, 30% w/w to 40% w/w, 30% w/w to 50% w/w, 35%w/w to 40% w/w, 35% w/w to 50% w/w, or 40% w/w to 50% w/w disaccharides.The thermochemically treated biomass may comprise less than 5% w/w, 10%w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, 35% w/w, 40% w/w, or 50% w/wdisaccharides.

II. Enzyme Treatment and Downstream Processing

The methods of the present disclosure may also include contacting, in asolution or suspension, one or more polysaccharide-cleaving enzymes andthe thermochemically treated biomass to form one or moreoligosaccharides. Furthermore, the methods may include enriching thesolution or suspension to increase the concentration of the one or moreoligosaccharides to form the ingredient. The one or moreoligosaccharides may be purified from the solution or suspension asprovided herein.

One or more steps of the method of forming or manufacturing thecomposition may be an enzymatic reaction, in which one or more enzymesare placed in a suitable reaction vessel together with one or morefeedstocks or biomasses (e.g., plant biomasses), which may be soluble orinsoluble in water, and a suitable solvent. As used herein, the term“plant biomass” may be replaced with the terms “feedstock” or “biomass”(e.g., a biomass not derived from a plant) unless indicated otherwise.

A variety of enzymes may be suitable for use in the enzymatic reaction.Any enzyme which produces oligosaccharides when acting on apolysaccharide-containing feedstock may be suitable. For example, theenzymatic reaction may comprise a cellulase, an endo-glucanase, acellobiohydrolase, a lytic polysaccharide monooxygenase (LPMO), alichenase, a xyloglucan endoglucanase (XEG), a mannanase, a chitinase, axylanase, and/or one or more suitable enzymes.

In various cases, the enzymatic reaction may comprise a cellulolyticpreparation from a species, such as Trichoderma reesei, which may bepurified and/or pretreated and/or may be supplemented with one or moreadditional enzymes, for example, adding a beta-glucanase, abeta-xylanase, and a cellobiohydrolase; a beta-glucanase, abeta-xylanase, an LPMO, and a cellobiohydrolase; an LPMO and a xylanase;or an LPMO, a xylanase, and a lichenase. Each enzyme may be provided tothe enzymatic reaction as a purified enzyme, a semi-purified mixturederived from some natural source or lab-grown culture, in the form of amicrobial strain engineered to produce the enzyme, or in any othersuitable manner. Fusions of these enzymes, either with other enzymes orwith non-enzymatic modules such as carbohydrate-binding modules (CBMs),are also envisaged. For example, an LPMO fused to a CBM, a xylanasefused to a CBM, or a xylanase fused to an LPMO may be utilized.

Aerobic conditions may be used for the one or more enzymatic reactions.Aerobic condition may comprise the addition of oxygen, which may beprovided by aeration of the substrate mixture with an oxygen-comprisinggas, such as air. Aeration may be conducted by the introduction ofoxygen-comprising air bubbles into the aqueous substrate mixtures byvarious systems, such as an air-injector, an aeration frit, a membranesystem, or an internal-loop airlift reactor. In some cases, theconcentration of molecular oxygen in the enzymatic reaction may be fromabout 4 mg/L to about 14 mg/L.

Another exemplary enzyme is a lichenase, which may be selected from theGH5, GH7, GH8, GH9, GH12, GH16, GH17, or GH26 families. For example,GH16 enzyme such as a GH16 enzyme derived from Bacillus subtilis may beutilized. The enzyme may be able to act on, for example, mixed-linkageglucans, which are glucans comprising a mixture of β-1,3 and β-1,4linkages, and may cleave them at β-1,4 glycosidic bonds. In the case inwhich the lichenase acts on a mixed-linkage glucan, the β-glucansproduced may fall largely within the size range of from 3 to about 7residues, so they may be particularly useful in the food, cosmetics, andnutraceutical industries. Mixed-linkage glucans are abundant in membersof the grass and horsetail families, and as such, grass-based feedstockssuch as straw generally have high levels of mixed-linkage glucans andmay be acted upon usefully with lichenases. The lichenases may includeGH5 lichenase from Bacillus subtilis.

Another alternative enzyme is a xylanase, which may act on, for example,feedstocks comprising a xylan backbone. The xylanase may be, forexample, a glucuronoxylanase, an arabinoxylanase, or aglucuronoarabinoxylanase. The enzyme may be active on a variety ofpolymers having a xylan backbone, such as glucuronoxylan, arabinoxylan,and glucuronoarabinoxylan. These polymers are generally abundant invarious plant-derived feedstocks, for example, both hardwood andsoftwood may comprise suitable polysaccharides, with hardwood oftencomprising glucuronoxylan and softwood often comprisingarabinoglucuronoxylan. Xylanases may include GH5 xylanases fromRuminiclostridium thermocellum and Gonapodya prolifera, and GH30xylanases from Dickeya chrysanthemi, Bacillus subtilis, Bacteroidesovatus, and Trichoderma reesei.

Another alternative enzyme is a mannanase, which may act on, forexample, feedstocks comprising a mannan backbone. The mannanase may be,for example, a mannanase, an glucomannanase, a galactomannanase or agalactoglucomannanase. The enzyme may be active on a variety of polymershaving a mannan backbone, such as mannan, glucomannan, galactomannan, orgalactoglucomannan. These polymers are generally abundant in variousplant-derived feedstocks, for example, both hardwood and softwood maycomprise appropriate polysaccharides. Suitable mannanases can includeGH5 mannanases from Trichoderma reesei and Aspergillus niger and a GH26mannanase from Aspergillus niger.

Other enzymes may include xyloglucanases and xyloglucan endoglucanases(XEGs), which are produced by numerous organisms, includingplant-pathogenic microbes. Xyloglucanases and XEGs may be able to act onxyloglucan, a hemicellulosic β-1,4 glucan chain abundant in the primarycell wall of higher plants, which is decorated with xylose, some of thexylose residues being further decorated with other residues, such asgalactose. When appropriate xyloglucanases or XEGs act on xyloglucan,the products may comprise xyloglucan oligosaccharides having a mainchain of a length useful in the foodstuff, cosmetics, and nutraceuticalindustries. Suitable xyloglucanases may include a GH5 xyloglucanase fromBacteroides ovatus and a GH74 xyloglucanase from Trichoderma reesei.

The enzymatic reaction may take place in solution and/or suspension. Theenzymatic reaction may take place in a suitable reaction vessel. In somecases, the enzymatic reaction may take place at a temperature ortemperature protocol suitable for the particular combination of enzymeand feedstock, the reaction may be allowed to progress for a certainamount of time (e.g., a predetermined amount of time) until the productshave reached a desired concentration or until some other requirement hasbeen met.

In order to ensure optimal contact between the enzymes and feedstock,the reaction mixture may be agitated, either constantly or at intervals.The agitation may take the form of (i) rhythmically moving the entirereaction vessel, (ii) a fan or other stirring device, (iii) a bubblesparging, or (iv) any other suitable method of agitation.

The enzymatic reaction may be a microbial fermentation. The temperatureand reaction time may be suitable for the growth of the microbialorganism used. The microbial organism may be genetically altered toproduce an enzyme suitable for the production of an oligosaccharidecomposition. The microbe may be a bacterium, for example, Escherichiacoli or a fungus, such as Saccharomyces cerevisiae or Trichodermareesei.

In some embodiments, an expression vector suitable for modifying thesubject microorganism may be used such that it produces an enzyme ormixture of enzymes as described elsewhere herein. Where desired, theexpression vector may be a plasmid or any other nucleic acid able toinduce production of the enzyme. In some instances, the expressionvector may comprise one or more of the following regulatory sequences soas to control the expression of the exogenous enzyme: regulatorysequences of a heat shock gene, regulatory sequences of a toxicity gene,regulatory sequences of a spore formation gene, or any other suitableregulatory sequence.

The enzymatic reaction can be carried out at a temperature ortemperature protocol suitable for the enzymes and substrates used. Forexample, the enzymatic reaction may be carried out at a constanttemperature in the range of from 10° C. to 100° C., from 20° C. to 80°C., or from 40° C. to 60° C. In some cases, if the enzymatic reactiontakes the form of a microbial fermentation, wherein the temperature maybe appropriate for such. For example, the enzymatic reaction maycomprise the growth of E. coli and/or the temperature may besubstantially constant and at about 37° C.

The pH of the solution or suspension may affect the activity of theenzymes. Control of pH may aid in assuring that an enzymatic reactionproceeds at a suitable rate. The enzymatic reaction may take place at apH in the range of from 2 to 10, 3 to 8, or 4 to 6.

The enzymatic reaction may be allowed to continue for a certain timeperiod before being quenched and the products isolated or otherwisecollected. This time period may be from 1 minute to 6 days, 0.5 days to5 days, or 16 hours to 96 hours. The reaction may alternatively beallowed to proceed until no further catalysis occurs.

The one or more feedstocks added to the enzymatic reaction may comprisepolysaccharides. Such polysaccharides may have been produced by aseparate reaction proceeding simultaneously, or substantiallysimultaneously, in the reaction vessel. The polysaccharides present inthe enzymatic reaction may be partially cleaved by enzymes into usefuloligosaccharides, leaving partially cleaved or uncleavedpolysaccharides, which may include, but are not limited to, cellulose,xylan (such as glucuronoxylan, arabinoxylan, or glucuronoarabinoxylan),mannan (such as glucomannan, galactomannan, or galactoglucomannan),mixed-linkage glucan, xyloglucan chitin, chitosan, or lignocellulose.

The enzymatic reaction may be allowed to continue to run until there isfrom 5% to 75%, 5% to 70%, 5% to 65%, 5% to 55%, or 10% to 50%undigested polysaccharide-containing feedstocks remaining. This can bemonitored or checked by reducing end assays, such as the anthrone assayand/or by chromatographic methods such as thin-layer chromatographyand/or high-performance anion exchange chromatography.

Any substance which comprises appropriate polysaccharides may form partof the feedstock. As the foodstuff, cosmetic, and nutraceuticalindustries generally use a broad variety of oligosaccharides, thepolysaccharides appropriate for taking part in the enzymatic reactionare not particularly limited. Feedstocks suitable for producing theoligosaccharide profile may comprise, for example, cellulose,lignocellulose, chitin, chitosan, xylan (such as glucuronoxylan,arabinoxylan, and glucuronoarabinoxylan) and/or mannan (such asglucomannan, galactomannan, or galactoglucomannan), however, anyfeedstock which can be suitably acted upon is envisaged. The feedstocksmay comprise sugar cane, corn stover, corncob, wheat bran, wheat straw,hardwood, softwood, or any other suitable biomass or plant biomass.

The feedstocks comprising such polysaccharides are also not particularlylimited, as most plant matter is rich in such polymers. As such, thefeedstock may comprise plant biomass such as grain, grain chaff, beanpods, seed coats, and/or other seed materials; seaweeds; corn stover,straw, bagasse, miscanthus, sorghum residue, switch grass, bamboo,and/or other monocotyledonous tissue; water hyacinth, leaf tissue,roots, and/or other vegetative matter; hardwood, hardwood chips,hardwood pulp, softwood, softwood chips, softwood pulp, paper, paperpulp, cardboard, and/or other wood-based feedstocks; crab shells, squidbiomass, shrimp shells, and/or other marine biomass, and/or anycombination of appropriate feedstocks. The feedstock may comprise wheatstraw or wood. As any given natural feedstock is likely to comprise amixture of different polysaccharides, it will sometimes be the case thata mixture of different enzymes is beneficial. Such a mixture maycomprise one or more of any suitable enzyme as discussed herein. Forexample, such a mixture might comprise an LPMO with an endo-glucanase, axylanase with a lichenase, a cellobiohydrolase with a mannanase, or anendo-glucanase with a cellobiohydrolase. In some embodiments, the enzymepartners may be present in molar ratios, for example, from 1:100 to100:1. In addition, as many appropriate feedstocks are recalcitrant,pretreatment of the feedstock is envisaged.

After the enzymatic reaction has progressed to a desired point, the oneor more oligosaccharides and the one or more polysaccharides from theenzymatic reaction mixture may be separated. This process can beperformed in a variety of ways depending on the composition of thebiomass used and the specificity of the enzymes used. As the reactionmixture will often comprise a mixture of soluble oligosaccharides andinsoluble polysaccharides, the reaction mixture may be filtered toremove insoluble matter and prepare the soluble oligosaccharide obtainedfor further processing.

The oligosaccharides may also be separated from the polysaccharides in anumber of ways. They may be isolated based on solubility, so that acomposition of soluble saccharides only is extracted for furtherprocessing, and/or isolated chromatographically to produce a compositionwith a narrower band of oligosaccharide chain lengths. Isolation may,for example, be based on precipitation, size-exclusion chromatography,ion-exchange chromatography, filtration, ultrafiltration,microfiltration, or nanofiltration. In the case that isolation based onsolubility is carried out, the profile of saccharides present in theisolated composition will generally depend on the original enzymaticreaction, as different polysaccharides generally decrease in solubilitywith length at different rates.

Also envisaged is the further treatment of all or part of the producedoligosaccharides to produce further products before incorporation into afoodstuff, cosmetic, or nutraceutical. This further treatment maycomprise any chemical, physical, or enzymatic step, such as reduction,for example, reductive amination where appropriate; oxidation,caramelization, modification with a Schiff base, or via the Maillardreaction, or by any combination of such steps, and may provide differentproducts having properties which are achieved or improved for thedesired purpose. For example, the caramelization properties, calorificvalue, flavor, and color may be modified. The oligosaccharides may alsobe purified, for example, through precipitation, size-exclusionchromatography, ion-exchange chromatography, filtration,ultrafiltration, microfiltration, or nanofiltration.

Also envisaged is the further treatment of all or part of the producedpolysaccharide fraction to produce products with improved propertiesbefore incorporation into a foodstuff, cosmetic, or nutraceutical. Thisfurther treatment may comprise any chemical, physical, or enzymaticstep, such as alkylation or acid-treatment. The polysaccharides may alsobe purified, for example, through precipitation, size-exclusionchromatography, ion-exchange chromatography, filtration,ultrafiltration, microfiltration, or nanofiltration.

In certain instances, following modification and/or purification of theoligosaccharide and polysaccharide fractions, all or part of thefractions can then be recombined at a ratio of from 1:100 to 1:1polysaccharide:oligosaccharide, for example, from 1:10 to 1:1, from 1:90to 1:2, from 1:80 to 1:3, from 1:70 to 1:4, or from 1:60 to 1:5. Thespecific ratio may depend on the desired properties of the finalingredient as well as the modifications and purifications that have beenapplied to the fractions. It may not be required to recombine all of theoligosaccharide and polysaccharide isolated from the enzymatic reaction.

The fractions can be recombined in a variety of ways, for example, bymixing a solution comprising all or part of the oligosaccharide fractionand a solution and/or suspension comprising all or part of thepolysaccharide fraction, which may further be dried, lyophilized, orcondensed in some other way. The fractions may also be recombined bymixing a dry form comprising all or part of the oligosaccharide fractionproduced by drying, lyophilization, or condensation in some other way,with a dry form comprising all or part of the polysaccharide fraction,produced by drying, lyophilization, or condensation in some other way.

The oligosaccharide components of the final composition may comprise oneor more of any type of oligosaccharide. For example, the oligosaccharidecomponents may comprise cello-oligosaccharides, xylo-oligosaccharides,mixed-linkage glucan oligosaccharides, manno-oligosaccharides,xyloglucan oligosaccharides, chito-oligosaccharides,arabinoxylo-oligosaccharides, or derivatives of any of theaforementioned oligosaccharides.

Any such dry or liquid composition may be deemed an ingredient suitablefor incorporation into a foodstuff, cosmetic, or nutraceutical at anystage of this process. This includes compositions that may be deemed tobe an intermediate during the method, such as a composition formed afterthe recombining of the oligosaccharide and polysaccharide fractionsprior to any further purification, optimization, drying, dissolving, orany other such steps, as well as including the final compositionobtained from the method.

As described herein, dry compositions may be formed by drying and/orlyophilization. The dry compositions can be dissolved into a solution ofvarious liquids including water, syrups, pastes, solvents, alcohols,etc. to form the liquid composition ingredient suitable forincorporation into a foodstuff, cosmetic, or nutraceutical. Liquidcompositions may be particularly useful in foods that require a smoothtexture such as candy, chocolate, and yogurts.

In some embodiments, following modification and/or purification of theoligosaccharide and polysaccharide fractions, all or part of thefractions may then be recombined at a ratio of from 1:100 to 1:1polysaccharide:oligosaccharide, for example, from 1:10 to 1:1, from 1:90to 1:2, from 1:80 to 1:3, from 1:70 to 1:4, or from 1:60 to 1:5. Thespecific ratio may depend on the desired properties of the finalingredient as well as the modifications and purifications that have beenapplied to the fractions.

Once a composition of the oligosaccharide products suitable for theapplication being considered is obtained, and further treatment and/orisolation can be carried out. The derivation of a foodstuff, cosmetic,or nutraceutical from the composition can furnish a broad array ofpotential uses. The ingredients as described herein, can be useful inapplications in which oligosaccharides, sugar, bulking sweeteners,low-intensity sweeteners, or other related food ingredients areconventionally used.

The polysaccharide-cleaving enzymes may be one of cellulase, xylanase,xyloglucanase, endo-glucanase, cellobiohydrolase, mannanase, lichenase,or a lytic polysaccharide monooxygenase (LPMO), for example, selectedfrom the group consisting of AA9, AA10, AA11, AA13, AA14, and AA15. Thepolysaccharide-cleaving enzyme may be prepared from T reesei fungiand/or the enzymatic reaction runs until there is 5-75%, 5-65%, or 5-50%undigested polysaccharide-containing feedstocks remaining.

The polysaccharide-cleaving enzymes may be operably linked to acatalytic or non-catalytic module, for example, wherein thepolysaccharide-cleaving enzyme may be operably linked to a non-catalyticmodule and the non-catalytic module is a carbohydrate-binding module.

In various embodiments, after the separating of the one or moreoligosaccharides and one or more polysaccharides, the one or moreoligosaccharides and one or more polysaccharides may be: purified;and/or undergo chemical, physical, or enzymatic treatment, such asreduction, oxidation, caramelization, or Maillard reaction; and/or maybe recombined by combining a dried powder of oligosaccharides with adried polysaccharide powder.

In some embodiments, the ingredient may comprise three or moreoligosaccharides of different molecular weights, wherein the method maycomprise forming the three or more oligosaccharides by an enzymaticreaction, wherein the enzymatic reaction comprises the step ofcontacting, in a solution or suspension, one or morepolysaccharide-cleaving enzymes and one or more feedstocks.

III. Foodstuff, Cosmetic, or Nutraceutical Ingredients Compositions

The polysaccharide components of the composition may comprise one ormore of any type of polysaccharide. For example, the polysaccharide maycomprise cellulose, lignocellulose, xylan, mixed-linkage glucan, mannan,xyloglucan, chitin, chitosan, or derivatives of any of theaforementioned polysaccharides.

The composition or ingredient may comprise various oligosaccharides. Thecomposition may include the oligosaccharides at varying amounts, forexample, depending on the desired properties of the composition. In someinstances, the composition may comprise at least 20% by dry weight, forexample, at least 30% by dry weight, cello-oligosaccharides having adegree of polymerization of from two to six; and/or the composition maycomprise at least 20% by dry weight, for example, at least 30% by dryweight, xylo-oligosaccharides having a degree of polymerization of fromtwo to twelve; and/or the composition may comprise at least 20% by dryweight, for example, at least 30% by dry weight, mixed-linkage glucanoligosaccharides having a degree of polymerization of from two to five;and/or the composition may comprise at least 20% by dry weight, forexample, at least 30% by dry weight, manno-oligosaccharides having adegree of polymerization of from two to twelve; and/or the compositionmay comprise at least 20% by dry weight, for example, at least 30% bydry weight, xyloglucan oligosaccharides having a degree ofpolymerization of from four to twelve, and/or the composition maycomprise at least 20% by dry weight, for example, at least 30% by dryweight, chito-oligosaccharides having a degree of polymerization of fromtwo to twelve; and/or the composition may comprise at least 20% by dryweight, for example, at least 30% by dry weight,arabinoxylo-oligosaccharides having a degree of polymerization of fromthree to fifteen. In certain embodiments, it may be understand that thecomposition can comprise a maximum of 100% by dry weight of the aboveoligosaccharides, therefore, the above embodiment, wherein theoligosaccharides are present in at least 20% by dry weight, does notcomprise all seven types of oligosaccharides.

In various embodiments, the composition or ingredient may comprise about5% to about 50% w/w cello-oligosaccharides with a degree ofpolymerization of from two to six. In certain embodiments, thecomposition or ingredient may comprise about 5% to about 50%, about 10%to about 40%, about 15% to about 35% w/w cello-oligosaccharides with adegree of polymerization of from two to six. The composition oringredient may comprise at least 5%, 8%, 10%, 15%, 20%, or 25% w/wcello-oligosaccharides with a degree of polymerization of from two tosix. In some embodiments, the composition or ingredient may compriseabout 20% to about 90% w/w cello-oligosaccharides with a degree ofpolymerization of from two to six. In certain embodiments, thecomposition or ingredient may comprise about 5% to about 95%, about 10%to about 92.5%, about 30% to about 80%, about 40% to about 70%, or about50% to about 60% w/w cello-oligosaccharides with a degree ofpolymerization of from two to six.

In various embodiments, the composition or ingredient may comprise about20% to about 90% w/w xylo-oligosaccharides with a degree ofpolymerization of from two to five. In certain embodiments, thecomposition or ingredient may comprise about 5% to about 95%, about 10%to about 92.5%, about 30% to about 80%, about 40% to about 70%, or about50% to about 60% w/w xylo-oligosaccharides with a degree ofpolymerization of from two to five. For example, the composition maycomprise at least 30% w/w of xylo-oligosaccharides with a degree ofpolymerization from two to five. The composition or ingredient maycomprise at least 5%, 8%, 10%, 15%, 20%, or 25% w/wxylo-oligosaccharides with a degree of polymerization of from two tofive.

In certain embodiments, the composition or ingredient may comprise about0.1% to about 15% w/w arabinoxylo-oligosaccharides with a degree ofpolymerization of from three to twelve. The composition or ingredientmay comprise at least 0.1%, 0.3%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w arabinoxylo-oligosaccharides with a degreeof polymerization of from three to twelve. In various embodiments, thecomposition or ingredient may comprise about 0.5% to about 25% w/warabinoxylo-oligosaccharides with a degree of polymerization of fromthree to fifteen. The composition or ingredient may comprise at least0.1%, 0.3%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, or35% w/w arabinoxylo-oligosaccharides with a degree of polymerization offrom three to fifteen.

In some embodiments, provided herein is the use of an oligosaccharidemixture in the formation of a foodstuff, cosmetic, or nutraceutical,wherein the oligosaccharide mixture comprises two oligosaccharidesselected from the list consisting of:

-   -   i) cello-oligosaccharides having a degree of polymerization of        from two to six;    -   ii) xylo-oligosaccharides having a degree of polymerization of        from two to twelve;    -   iii) mixed-linkage glucan oligosaccharides having a degree of        polymerization of from two to five;    -   iv) manno-oligosaccharides having a degree of polymerization of        from two to twelve;    -   v) xyloglucan oligosaccharides having a degree of polymerization        of from four to ten;    -   vi) chito-oligosaccharides having a degree of polymerization of        from two to twelve; and/or    -   vii) arabinoxylo-oligosaccharide having a degree of        polymerization of from three to fifteen, wherein the two        oligosaccharides may be present in a ratio of from 1:9 to 9:1,        1:4 to 4:1, or 2:3 to 3:2 in relation to each other.

In certain cases, the arabinoxylo-oligosaccharides may comprise at least0.1% arabinosyl residues. The arabinoxylo-oligosaccharides may compriseat least 0.1%, 0.2%, 0.5%, 1%, 5%, or 10% arabinosyl residues.

The amounts of each of the oligosaccharides may be varied depending onthe desired properties of the resulting foodstuff, cosmetic, ornutraceutical. For example, the two oligosaccharides may be present in aratio of 1:9 to 1:1, 1:2 to 1:1, or 2:3 to 1:1 in relation to eachother.

The oligosaccharide mixture may further comprise a thirdoligosaccharide. The oligosaccharide mixture may comprise a thirdoligosaccharide and a fourth oligosaccharide. The oligosaccharidemixture may comprise a third oligosaccharide, a fourth oligosaccharide,and a fifth oligosaccharide. The oligosaccharide mixture may furthercomprise a third oligosaccharide, a fourth oligosaccharide, a fiftholigosaccharide, and a sixth oligosaccharide. The oligosaccharidemixture may further comprise a third oligosaccharide, a fourtholigosaccharide, a fifth oligosaccharide, a sixth oligosaccharide, and aseventh oligosaccharide. These oligosaccharides may be selected from thesame list as the at least two oligosaccharides as provided above.

Oligosaccharide mixtures of the at least two oligosaccharides maycomprise the cello-oligosaccharides, for instance,cello-oligosaccharides in combination with the xylo-oligosaccharides. Analternative composition may comprise cello-oligosaccharides incombination with manno-oligosaccharides. In some embodiments, theoligosaccharide mixtures may include cello-oligosaccharides,xylo-oligosaccharides, and arabinoxylo-oligosaccharides in combinationwith each other.

The oligosaccharide mixtures of the at least two oligosaccharides mayadditionally include a polysaccharide, for example, a cellulosicpolysaccharide, such as cellulose, or a polysaccharide derivative, forexample, a cellulose derivative, such as carboxymethylcellulose, or apolysaccharide aggregate, for example, a portion of lignocellulosicbiomass. In some instances, the ratio in the combination may be from1:100 to 1:1 polysaccharide/polysaccharide derivative/polysaccharideaggregate:oligosaccharide, for example, from 1:90 to 1:2, from 1:80 to1:3, from 1:70 to 1:4, or from 1:60 to 1:5. As such, the ratio betweenthe first oligosaccharide, the second oligosaccharide, and thepolysaccharide may be from 2:2:1 to 30:30:1, for example, about 3:3:1.

Combinations of Oligosaccharides

A composition may comprise a mixture of one or more oligosaccharides. Amixture of oligosaccharides may comprise two forms or types ofoligosaccharides, for instance, cello-oligosaccharides andxylo-oligosaccharides. A mixture of oligosaccharides may comprise threeforms of oligosaccharides, for instance, cello-oligosaccharides,manno-oligosaccharides, and xylo-oligosaccharides. A mixture ofoligosaccharides may comprise four forms of oligosaccharides, forinstance, cello-oligosaccharides, manno-oligosaccharides, mixed-linkageglucan oligosaccharides, and chito-oligosaccharides.

An oligosaccharide mixture may comprise two forms of oligosaccharides,for example, a first oligosaccharide and a second oligosaccharide. Anoligosaccharide mixture may comprise about 5% of a first oligosaccharideand about 95% of a second oligosaccharide w/w. An oligosaccharidemixture may comprise about 10% of a first oligosaccharide and about 90%of a second oligosaccharide w/w. An oligosaccharide mixture may compriseabout 15% of a first oligosaccharide and about 85% of a secondoligosaccharide w/w. An oligosaccharide mixture may comprise about 20%of a first oligosaccharide and about 80% of a second oligosaccharidew/w. An oligosaccharide mixture may comprise about 25% of a firstoligosaccharide and about 75% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise about 30% of a firstoligosaccharide and about 70% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise about 35% of a firstoligosaccharide and about 65% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise about 40% of a firstoligosaccharide and about 50% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise 45% of a first oligosaccharide and55% of a second oligosaccharide w/w. An oligosaccharide mixture maycomprise 50% of a first oligosaccharide and 50% of a secondoligosaccharide w/w. An oligosaccharide mixture may comprise 55% of afirst oligosaccharide and 45% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise 60% of a first oligosaccharide and30% of a second oligosaccharide w/w. An oligosaccharide mixture maycomprise 65% of a first oligosaccharide and 35% of a secondoligosaccharide w/w. An oligosaccharide mixture may comprise 70% of afirst oligosaccharide and 30% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise 75% of a first oligosaccharide and25% of a second oligosaccharide w/w. An oligosaccharide mixture maycomprise 80% of a first oligosaccharide and 20% of a secondoligosaccharide w/w. An oligosaccharide mixture may comprise 85% of afirst oligosaccharide and 15% of a second oligosaccharide w/w. Anoligosaccharide mixture may comprise 90% of a first oligosaccharide and10% of a second oligosaccharide w/w. An oligosaccharide mixture maycomprise 95% of a first oligosaccharide and 5% of a secondoligosaccharide w/w. In some cases, a first oligosaccharide may becello-oligosaccharides and a second oligosaccharide may bexylo-oligosaccharides. In some instances, a first oligosaccharide may becello-oligosaccharides and a second oligosaccharide may bemanno-oligosaccharides. In some embodiments, a first oligosaccharide maybe xylo-oligosaccharides and a second oligosaccharide may bemanno-oligosaccharides. Other combinations of a first oligosaccharideand a second oligosaccharide are also within the scope of thisdisclosure.

An oligosaccharide mixture may comprise three forms of oligosaccharides,for example a first oligosaccharide, a second oligosaccharide, and athird oligosaccharide. An oligosaccharide mixture may comprise about 20%of a first oligosaccharide, 40% of a second oligosaccharide, and 40% ofa third oligosaccharide w/w. An oligosaccharide mixture may compriseabout 30% of a first oligosaccharide, 30% of a second oligosaccharide,and 40% of a third oligosaccharide w/w. An oligosaccharide mixture maycomprise about 10% of a first oligosaccharide, 10% of a secondoligosaccharide, and 80% of a third oligosaccharide w/w. Anoligosaccharide mixture may comprise about 20% of a firstoligosaccharide, 20% of a second oligosaccharide, and 60% of a thirdoligosaccharide w/w. An oligosaccharide mixture may comprise about 20%of a first oligosaccharide, 30% of a second oligosaccharide, and 50% ofa third oligosaccharide w/w. In some examples, a first oligosaccharidemay be manno-oligosaccharides, a second oligosaccharide may bexylo-oligosaccharides, and a third oligosaccharide may becello-oligosaccharides. In some examples, a first oligosaccharide may bexyloglucan-oligosaccharides, a second oligosaccharide may bexylo-oligosaccharides, and a third oligosaccharide may becello-oligosaccharides. Other combinations of a first oligosaccharide, asecond oligosaccharide, and a third oligosaccharide are also within thescope of this disclosure.

An oligosaccharide mixture may comprise two or more oligosaccharides, afirst oligosaccharide and a second oligosaccharide which is differentthan the first oligosaccharide. For instance, the first oligosaccharidemay be a xylo-oligosaccharide or a cello-oligosaccharide or amanno-oligosaccharide or other oligosaccharide as provided herein,whereas the second oligosaccharide can be a xylo-oligosaccharide or acello-oligosaccharide or a manno-oligosaccharide or otheroligosaccharides not used as the first oligosaccharide. Stated anotherway, the first oligosaccharide can be different than the secondoligosaccharide (e.g., the first oligosaccharide can be of a differenttype of oligosaccharide than the second oligosaccharide). The ratio of afirst oligosaccharide to a second oligosaccharide in the mixture may beabout 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9.

The ratio of a first oligosaccharide to a second oligosaccharide in themixture may be about 2:1, 2:3, 2:5, 2:7, or 2:9. The oligosaccharidesmay be cello-oligosaccharides, manno-oligosaccharides,xylo-oligosaccharides, xyloglucan-oligosaccharides, mixed-linkageoligosaccharides, chito-oligosaccharides, arabinoxylo-oligosaccharides,or other oligosaccharides as provided herein, wherein the firstoligosaccharide is selected to be a different oligosaccharide than thesecond oligosaccharide. In other words, the first oligosaccharide may bea different type of oligosaccharide than the second oligosaccharide.

The ratio of a first oligosaccharide to a second oligosaccharide in themixture may be about 3:1, 3:2, 3:4, 3:5, 3:7, or 3:8. Theoligosaccharides may be cello-oligosaccharides, manno-oligosaccharides,xylo-oligosaccharides, xyloglucan-oligosaccharides, mixed-linkageoligosaccharides, chito-oligosaccharides, arabinoxylo-oligosaccharides,or other oligosaccharides provided herein, wherein the firstoligosaccharide is selected to be a different oligosaccharide than thesecond oligosaccharide.

The ratio of a first oligosaccharide to a second oligosaccharide in anoligosaccharide mixture comprising two or more oligosaccharides may befrom 1:9 to 9:1, from 1:4 to 4:1, from 1:3 to 3:1, or from 2:3 to 3:2.The oligosaccharides may be cello-oligosaccharides,manno-oligosaccharides, xylo-oligosaccharides,xyloglucan-oligosaccharides, mixed-linkage oligosaccharides,chito-oligosaccharides, arabinoxylo-oligosaccharide, or otheroligosaccharides provided herein, wherein the first oligosaccharide isselected to be a different oligosaccharide than the secondoligosaccharide.

In some cases, the composition or the ingredient may include at least 1%w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w,30% w/w, or more of cellobiose, xylobiose, mannobiose (e.g.,Man-β-1,4-Man), Glc-β-1,4-Man, Man-β-1,4-Glc, laminaribiose,gentiobiose, sophorose, maltose, lactose, or sucrose. In certain cases,the composition or the ingredient may include at least 1% w/w, 2% w/w,3% w/w, 4% w/w, 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, ormore of cellotriose, xylotriose, monoarabinosylated xylobiose,monoglucuronosylated xylobiose, maltotriose, mannotriose (e.g.,Man-β-1,4-Man-β-1,4-Man), Glc-β-1,4-Man-β-1,4-Man,Man-β-1,4-Glc-β-1,4-Man, Man-β-1,4-Man-β-1,4-Glc,Man-β-1,4-Glc-β-1,4-Glc, Glc-β-1,4-Man-β-1,4-Glc,Glc-β-1,4-Glc-β-1,4-Man, Glc-β-1,3-Glc-β-1,4-Glc, orGlc-β-1,4-Glc-β-1,3-Glc. In certain instances, the composition or theingredient may include at least 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w,10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, or more of xylotetraose,cellotetraose, monoarabinosylated xylotriose, monoglucuronosylatedxylotriose, diarabinosylated xylobiose, diglucuronosylated xylobiose,maltotetraose, mannotetraose (e.g., Man-β-1,4-Man-β-1,4-Man-β-1,4-Man),Glc-β-1,4-Man-β-1,4-Man-β-1,4-Man, Man-β-1,4-Glc-β-1,4-Man-β-1,4-Man,Man-β-1,4-Man-β-1,4-Glc-β-1,4-Man, Man-β-1,4-Man-β-1,4-Man-β-1,4-Glc,Glc-β-1,4-Glc-β-1,4-Man-β-1,4-Man, Man-β-1,4-Glc-β-1,4-Glc-β-1,4-Man,Man-β-1,4-Man-β-1,4-Glc-β-1,4-Glc, Glc-β-1,4-Man-β-1,4-Glc-β-1,4-Man,Glc-β-1,4-Man-β-1,4-Man-β-1,4-Glc, Man-β-1,4-Glc-β-1,4-Man-β-1,4-Glc,Glc-β-1,3-Glc-β-1,4-Glc-1,4-Glc, Glc-β-1,4-Glc-β-1,3-Glc-1,4-Glc,Glc-β-1,4-Glc-β-1,4-Glc-1,3-Glc, or Glc-β-1,3-Glc-β-1,4-Glc-1,3-Glc. Incertain cases, the composition or the ingredient may include at least0.01% w/w, 0.05% w/w, 0.1% w/w, 0.5% w/w, 1% w/w, 2% w/w, 5% w/w, 10%w/w, 15% w/w, 20% w/w, or more of xylopentaose, cellopentaose,monoarabinosylated xylotetraose, monoglucuronosylated xylotetraose,diarabinosylated xylotriose, diglucuronosylated xylotriose,maltopentaose, mannopentaose (e.g.,Man-β-1,4-Man-β-1,4-Man-β-1,4-Man-β-1,4-Man), mixed-linkageglucan-derived pentasaccharide, or mannan-derived pentasaccharide

The composition or ingredient may comprise from 1% to 50%, 5% to 40% 10%to 30%, or 15% to 25% w/w of cellobiose. The composition or ingredientmay comprise from 2.5% to 90%, 5% to 80% 10% to 70%, or 20% to 60% w/wof xylobiose. The composition or ingredient may comprise from 2.5% to75%, 5% to 50% 10% to 40%, or 20% to 30% w/w of xylotriose.

Oligosaccharide Compositions with Varying Degrees of Polymerization

The average degree of polymerization of the oligosaccharides in thecomposition may be from 1 to 50, 1.5 to 25, 2 to 15, 2.1 to 10, 2.1 to7, or 2.2 to 5.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of two in a xylo-oligosaccharide mixture may be fromabout 2% to about 80% w/w. The concentration of xylo-oligosaccharideswith a degree of polymerization of two may be at least 2%, 4%, 6%, 8%,10%, 12%, 15%, 18%, 20%, 25%, or 30% w/w. The concentration ofxylo-oligosaccharides with a degree of polymerization of two may behigher in some cases, for instance, up to 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of three in a xylo-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of three may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, or 20% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of four in a xylo-oligosaccharide mixture may be about 5%to about 20% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of four may be at least 5%, 8%, 10%, 12%, 15%,18%, or 20% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of five in a xylo-oligosaccharide mixture may be about 5%to about 20% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of five may be at least 5%, 7%, 8%, 10%, 12%,15%, 18%, or 20% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of six in a xylo-oligosaccharide mixture may be about 5%to about 25% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of six may be at least 5%, 8%, 10%, 12%, 15%,18%, 20%, or 25% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of seven in a xylo-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of seven may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 17%, or 20% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of eight in a xylo-oligosaccharide mixture may be about1% to about 15% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of eight may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of nine in a xylo-oligosaccharide mixture may be about 2%to about 15% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of nine may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of ten in a xylo-oligosaccharide mixture may be about 2%to about 15% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of ten may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of eleven in a xylo-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of eleven may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xylo-oligosaccharides with a degree ofpolymerization of twelve in a xylo-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of xylo-oligosaccharides with adegree of polymerization of twelve may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of cello-oligosaccharides with a degree ofpolymerization of two in a cello-oligosaccharide mixture may be about 2%to about 80% w/w. The concentration of cello-oligosaccharides with adegree of polymerization of two may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, 20%, 25%, or 30% w/w. The concentration ofcello-oligosaccharides with a degree of polymerization of two may behigher in some cases, for instance, at least 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, or 80% w/w.

The concentration of cello-oligosaccharides with a degree ofpolymerization of three in a cello-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of cello-oligosaccharides with adegree of polymerization of three may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, or 20% w/w.

The concentration of cello-oligosaccharides with a degree ofpolymerization of four in a cello-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of cello-oligosaccharides with adegree of polymerization of four may be at least 5%, 8%, 10%, 12%, 15%,18%, or 20% w/w.

The concentration of cello-oligosaccharides with a degree ofpolymerization of five in a cello-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of cello-oligosaccharides with adegree of polymerization of five may be at least 5%, 7%, 8%, 10%, 12%,15%, 18%, or 20% w/w.

The concentration of cello-oligosaccharides with a degree ofpolymerization of six in a cello-oligosaccharide mixture may be about 5%to about 25% w/w. The concentration of cello-oligosaccharides with adegree of polymerization of six may be at least 5%, 8%, 10%, 12%, 15%,18%, 20%, or 25% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of two in a manno-oligosaccharide mixture may be about 2%to about 30% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of two may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, 20%, 25%, or 30% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of three in a manno-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of three may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, or 20% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of four in a manno-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of four may be at least 5%, 8%, 10%, 12%, 15%,18%, or 20% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of five in a manno-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of five may be at least 5%, 7%, 8%, 10%, 12%,15%, 18%, or 20% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of six in a manno-oligosaccharide mixture may be about 5%to about 25% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of six may be at least 5%, 8%, 10%, 12%, 15%,18%, 20%, or 25% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of seven in a manno-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of seven may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 17%, or 20% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of eight in a manno-oligosaccharide mixture may be about1% to about 15% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of eight may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of nine in a manno-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of nine may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of ten in a manno-oligosaccharide mixture may be about 2%to about 15% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of ten may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of eleven in a manno-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of eleven may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of manno-oligosaccharides with a degree ofpolymerization of twelve in a manno-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of manno-oligosaccharides with adegree of polymerization of twelve may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of four in a xyloglucan-oligosaccharide mixture may beabout 5% to about 20% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of four maybe at least 5%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of five in a xyloglucan-oligosaccharide mixture may beabout 5% to about 20% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of five maybe at least 5%, 7%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of six in a xyloglucan-oligosaccharide mixture may beabout 5% to about 25% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of six maybe at least 5%, 8%, 10%, 12%, 15%, 18%, 20%, or 25% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of seven in a xyloglucan-oligosaccharide mixture may beabout 2% to about 20% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of seven maybe at least 2%, 4%, 6%, 8%, 10%, 12%, 15%, 17%, or 20% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of eight in a xyloglucan-oligosaccharide mixture may beabout 1% to about 15% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of eight maybe at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of nine in a xyloglucan-oligosaccharide mixture may beabout 2% to about 15% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of nine maybe at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of xyloglucan-oligosaccharides with a degree ofpolymerization of ten in a xyloglucan-oligosaccharide mixture may beabout 2% to about 15% w/w. The concentration ofxyloglucan-oligosaccharides with a degree of polymerization of ten maybe at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of mixed-linkage glucan-oligosaccharides with a degreeof polymerization of two in a mixed-linkage glucan-oligosaccharidemixture may be about 2% to about 30% w/w. The concentration ofmixed-linkage glucan-oligosaccharides with a degree of polymerization oftwo may be at least 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, 20%, 25%, or 30%w/w.

The concentration of mixed-linkage glucan-oligosaccharides with a degreeof polymerization of three in a mixed-linkage glucan-oligosaccharidemixture may be about 2% to about 20% w/w. The concentration ofmixed-linkage glucan-oligosaccharides with a degree of polymerization ofthree may be at least 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of mixed-linkage glucan-oligosaccharides with a degreeof polymerization of four in a mixed-linkage glucan-oligosaccharidemixture may be about 5% to about 20% w/w. The concentration ofmixed-linkage glucan-oligosaccharides with a degree of polymerization offour may be at least 5%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of mixed-linkage glucan-oligosaccharides with a degreeof polymerization of five in a mixed-linkage glucan-oligosaccharidemixture may be about 5% to about 20% w/w. The concentration ofmixed-linkage glucan-oligosaccharides with a degree of polymerization offive may be at least 5%, 7%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of two in a chito-oligosaccharide mixture may be about 2%to about 30% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of two may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, 20%, 25%, or 30% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of three in a chito-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of three may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 18%, or 20% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of four in a chito-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of four may be at least 5%, 8%, 10%, 12%, 15%,18%, or 20% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of five in a chito-oligosaccharide mixture may be about5% to about 20% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of five may be at least 5%, 7%, 8%, 10%, 12%,15%, 18%, or 20% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of six in a chito-oligosaccharide mixture may be about 5%to about 25% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of six may be at least 5%, 8%, 10%, 12%, 15%,18%, 20%, or 25% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of seven in a chito-oligosaccharide mixture may be about2% to about 20% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of seven may be at least 2%, 4%, 6%, 8%, 10%,12%, 15%, 17%, or 20% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of eight in a chito-oligosaccharide mixture may be about1% to about 15% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of eight may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of nine in a chito-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of nine may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of ten in a chito-oligosaccharide mixture may be about 2%to about 15% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of ten may be at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10% or 15% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of eleven in a chito-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of eleven may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of chito-oligosaccharides with a degree ofpolymerization of twelve in a chito-oligosaccharide mixture may be about2% to about 15% w/w. The concentration of chito-oligosaccharides with adegree of polymerization of twelve may be at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of three in an arabinoxylo-oligosaccharide mixture may beabout 2% to about 20% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of threemay be at least 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of four in an arabinoxylo-oligosaccharide mixture may beabout 5% to about 20% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of four maybe at least 5%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of five in an arabinoxylo-oligosaccharide mixture may beabout 5% to about 20% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of five maybe at least 5%, 7%, 8%, 10%, 12%, 15%, 18%, or 20% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of six in an arabinoxylo-oligosaccharide mixture may beabout 5% to about 25% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of six maybe at least 5%, 8%, 10%, 12%, 15%, 18%, 20%, or 25% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of seven in an arabinoxylo-oligosaccharide mixture may beabout 2% to about 20% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of sevenmay be at least 2%, 4%, 6%, 8%, 10%, 12%, 15%, 17%, or 20% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of eight in an arabinoxylo-oligosaccharide mixture may beabout 1% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of eightmay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of nine in an arabinoxylo-oligosaccharide mixture may beabout 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of nine maybe at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of ten in an arabinoxylo-oligosaccharide mixture may beabout 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of ten maybe at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of eleven in an arabinoxylo-oligosaccharide mixture maybe about 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of elevenmay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of twelve in an arabinoxylo-oligosaccharide mixture maybe about 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of twelvemay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of thirteen in an arabinoxylo-oligosaccharide mixture maybe about 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of thirteenmay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of fourteen in an arabinoxylo-oligosaccharide mixture maybe about 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of fourteenmay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of fifteen in an arabinoxylo-oligosaccharide mixture maybe about 2% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of fifteenmay be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% w/w.

The concentration of arabinoxylo-oligosaccharides with a degree ofpolymerization of from three to twelve in an arabinoxylo-oligosaccharidemixture may be about 0.1% to about 15% w/w. The concentration ofarabinoxylo-oligosaccharides with a degree of polymerization of three totwelve may be at least 0.1%, 0.3%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, or 15% w/w.

Compositions with Combinations of Monosaccharides, Polysaccharides,and/or Oligosaccharides

In some embodiments, the composition or ingredient (e.g., the ingredientfor human consumption) may soluble in water. The solubility of theingredient in water may be at least 80 g of the ingredient per 100 g ofwater at 50° C.

The ingredient may be combined with a liquid to form a liquidingredient. In some cases, a viscosity of the liquid ingredient may becomparable or similar to a viscosity of corn syrup. In some other cases,that viscosity of the liquid ingredient may comparable or similar to aviscosity of high-fructose corn syrup. For example, the liquidingredient may have a viscosity of from 5 cps to 100,000 cps, 8,000 cpsto 100,000 cps, 10,000 cps to 50,000 cps, or 15,000 cps to 25,000 cps.Moreover, the liquid ingredient may have fewer calories per gram thancorn syrup or high-fructose corn syrup. The liquid ingredient may have alower glycemic index than corn syrup or high-fructose corn syrup.

In some embodiments, the liquid may comprise water or any other suitableliquid. The liquid ingredient may comprises at least 5%, 10%, 20%, 30%,40%, or 50% by dry weight of the at least one oligosaccharide.Furthermore, the liquid ingredient may comprise at least 0.2%, 0.5%, 1%,2%, 3%, 5%, or 10% by dry weight of the at least one polysaccharide. Forexample, the liquid ingredient may comprises at least 20% by dry weightof the at least one oligosaccharide and at least 2% by dry weight of theat least one polysaccharide. Other combinations of the at least oneoligosaccharide and the at least one polysaccharide are also within thescope of the present disclosure.

The liquid ingredient comprises at least 0.2%, 0.5%, 1%, 2%, 3%, 5%, or10% by dry weight of xylan. The liquid ingredient may comprise at least0.2%, 0.5%, 1%, 2%, 3%, 5%, or 10% by dry weight of mannan. The liquidingredient may comprise at least 0.2%, 0.5%, 1%, 2%, 3%, 5%, or 10% bydry weight of a cellulose derivative.

In various cases, the liquid ingredient may have a concentration ofpolysaccharides of from 0.1% to 50%, 0.1% to 40%, 0.1% to 30%, 0.1% to20%, 0.1% to 10%, 0.5% to 50%, or 1% to 50% w/v. For example, the liquidingredient may have a concentration of polysaccharides of from 0.1% to50% w/v. The liquid ingredient may comprise an amount of polysaccharideand oligosaccharide in a ratio from 1:200 to 1:1, 1:150 to 1:1, 1:125 to1:1 1:100 to 1:1, 1:90 to 1:1, 1:80 to 1:1, 1:70 to 1:1, 1:60 and 1:1,1:50 and 1:1, 1:25 and 1:1, or 1:10 and 1:1. For example, the liquidingredient may comprises an amount of polysaccharide and oligosaccharidein a ratio from 1:100 to 1:1.

The one or more soluble polysaccharides may comprise at least one of amannan, a xylan, a mixed-linkage glucan, a lignocellulose, ahemicellulose, a cellulose derivative, a chitosan, a xyloglucan, or anyother suitable soluble polysaccharide. The cellulose derivative maycomprise at least one of a cellulose acetate, a hydroxyethylcellulose, ahydroxymethylcellulose, or any other suitable cellulose derivative.

The biomass may comprise at least one of a sugar cane biomass, a cornbiomass, a wheat biomass, a hardwood biomass, a softwood biomass, or anyother suitable biomass.

In certain instances, a composition for human consumption may include asoluble polysaccharide and an oligosaccharide comprising at least one of(i) a cello-oligosaccharide having a degree of polymerization (DP) offrom two to six; (ii) a xylo-oligosaccharide having a DP of from two totwelve; (iii) a manno-oligosaccharide having a DP of from two to twelve;(iv) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; (v) a mixed-linkage glucan oligosaccharide having a DP of fromtwo to five; or (vi) a chito-oligosaccharide having a DP of from two totwelve. The composition may include less than 5% by dry weight solublepolysaccharides. In some cases, the composition may include less than1%, 2%, 5%, 7.5%, 10%, or 20% by dry weight soluble polysaccharides. Insome embodiments, the composition may be free, or substantially free, ofinsoluble polysaccharides.

A composition may comprise a combination of polysaccharides andoligosaccharides. In some embodiments, a composition may comprise acombination of oligosaccharides and soluble polysaccharides. The sourceof the polysaccharides (or the soluble polysaccharides) in suchcompositions may contain cellulose, such as biomass, for example, theundigested component of partially digested biomass, such as theundigested biomass from the same reaction as that which produced theoligosaccharides. The polysaccharides in the undigested biomass maycomprise lignin, polyphenol, cellulose, lignocellulose, or any othersuitable polysaccharides as described herein. Addition ofpolysaccharides (e.g., soluble polysaccharides) to oligosaccharidemixtures can be done to improve the gastrointestinal tolerance of theoligosaccharide mixtures. Oligosaccharide consumption can causegastrointestinal distress, including diarrhea, discomfort, and bloating.The compositions described herein may have an improved gastrointestinaltolerance such as, less or no discomfort, bloating, diarrhea, orgastrointestinal distress as compared to a saccharide compositionavailable commercially or a saccharide composition comprising primarilymonosaccharides and/or disaccharides. For example, a subject who ingestsone or more of the compositions provided herein may have an improvedgastrointestinal tolerance such as, less or no discomfort, bloating,diarrhea, or gastrointestinal distress as compared to if, or when, thesubject ingests a saccharide composition available commercially or asaccharide composition comprising primarily monosaccharides and/ordisaccharides.

The concentration of undigested biomass in a composition may be from 1%to 50% w/w. The concentration of undigested biomass in a composition maybe from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%,1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%, 5% to 15%, 5% to20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to 45%, 5% to 50%,10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%,10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%,15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to 30%, 20% to 35%,20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to 35%, 25% to 40%,25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to 45%, 30% to 50%,35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to 50%, or 45% to50% w/w. The concentration of undigested biomass in a composition may beabout 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w. Theconcentration of undigested biomass in a composition may be at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w. The concentration ofundigested biomass in a composition may be at most 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, or 50% w/w.

The concentration of soluble polysaccharides in a composition may befrom 1% to 50% w/w. The concentration of soluble polysaccharides in acomposition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%,5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to50%, or 45% to 50% w/w. The concentration of soluble polysaccharides ina composition may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or 50% w/w. The concentration of soluble polysaccharides in acomposition may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 45% w/w. The concentration of soluble polysaccharides in acomposition may be at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% w/w.

The concentration of xylo-oligosaccharides in a composition may be from1% to 50% w/w. The concentration of xylo-oligosaccharides in acomposition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%,5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to50%, or 45% to 50% w/w. The concentration of xylo-oligosaccharides in acomposition may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% w/w. The concentration of xylo-oligosaccharides in a compositionmay be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w.The concentration of xylo-oligosaccharides in a composition may be atmost 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

The concentration of cello-oligosaccharides in a composition may be from1% to 50% w/w. The concentration of cello-oligosaccharides in acomposition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%,5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to50%, or 45% to 50% w/w. The concentration of cello-oligosaccharides in acomposition may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% w/w. The concentration of cello-oligosaccharides in a compositionmay be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w.The concentration of cello-oligosaccharides in a composition may be atmost 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

In some embodiments, the composition may comprise at least 5% w/w ofcello-oligosaccharides and at least 5% w/w of a second oligosaccharides(e.g., at least 5% w/w of xylo-oligosaccharides, manno-oligosaccharides,mixed-linkage glucan oligosaccharides, xyloglucan-oligosaccharides,chito-oligosaccharides, arabinoxylo-oligosaccharides, or any othersuitable oligosaccharides).

The concentration of manno-oligosaccharides in a composition may be from1% to 50% w/w. The concentration of manno-oligosaccharides in acomposition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%,5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to50%, or 45% to 50% w/w. The concentration of manno-oligosaccharides in acomposition may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% w/w. The concentration of manno-oligosaccharides in a compositionmay be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w.The concentration of manno-oligosaccharides in a composition may be atmost 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

The concentration of chito-oligosaccharides in a composition may be from1% to 50% w/w. The concentration of chito-oligosaccharides in acomposition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to 10%,5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, 20% to30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to50%, or 45% to 50% w/w. The concentration of chito-oligosaccharides in acomposition may be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% w/w. The concentration of chito-oligosaccharides in a compositionmay be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w.The concentration of chito-oligosaccharides in a composition may be atmost 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

The concentration of xyloglucan-oligosaccharides in a composition may befrom 1% to 50% w/w. The concentration of xyloglucan-oligosaccharides ina composition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1%to 25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%,5% to 45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%,10% to 35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%,15% to 30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%,20% to 30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%,25% to 35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%,30% to 45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%,40% to 50%, or 45% to 50% w/w. The concentration ofxyloglucan-oligosaccharides in a composition may be about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w. The concentration ofxyloglucan-oligosaccharides in a composition may be at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w. The concentration ofxyloglucan-oligosaccharides in a composition may be at most 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

The concentration of mixed-linkage glucan-oligosaccharides in acomposition may be from 1% to 50% w/w. The concentration ofmixed-linkage glucan-oligosaccharides in a composition may be from 1% to5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%, 1% to 35%, 1%to 40%, 1% to 45%, 1% to 50%, 5% to 10%, 5% to 15%, 5% to 20%, 5% to25%, 5% to 30%, 5% to 35%, 5% to 40%, 5% to 45%, 5% to 50%, 10% to 15%,10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 10% to 45%,10% to 50%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%,15% to 45%, 15% to 50%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%,20% to 45%, 20% to 50%, 25% to 30%, 25% to 35%, 25% to 40%, 25% to 45%,25% to 50%, 30% to 35%, 30% to 40%, 30% to 45%, 30% to 50%, 35% to 40%,35% to 45%, 35% to 50%, 40% to 45%, 40% to 50%, or 45% to 50% w/w. Theconcentration of mixed-linkage glucan-oligosaccharides in a compositionmay be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.The concentration of mixed-linkage glucan-oligosaccharides in acomposition may be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 45% w/w. The concentration of mixed-linkage glucan-oligosaccharidesin a composition may be at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, or 50% w/w.

The concentration of arabinoxylo-oligosaccharides in a composition maybe from 1% to 50% w/w. The concentration of arabinoxylo-oligosaccharidesin a composition may be from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%,1% to 25%, 1% to 30%, 1% to 35%, 1% to 40%, 1% to 45%, 1% to 50%, 5% to10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%,5% to 45%, 5% to 50%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%,10% to 35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%,15% to 30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%,20% to 30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%,25% to 35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%,30% to 45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%,40% to 50%, or 45% to 50% w/w. The concentration ofarabinoxylo-oligosaccharides in a composition may be about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w. The concentration ofarabinoxylo-oligosaccharides in a composition may be at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% w/w. The concentration ofarabinoxylo-oligosaccharides in a composition may be at most 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.

A composition may comprise one or more polysaccharides (e.g., one ormore soluble polysaccharides) and one or more oligosaccharides. Thecomposition may comprise a polysaccharide and one type ofoligosaccharide. The composition may comprise a polysaccharide orplurality of polysaccharides and two forms of oligosaccharides. Thecomposition may comprise a polysaccharide or plurality ofpolysaccharides and three forms of oligosaccharides. The composition maycomprise a polysaccharide or plurality of polysaccharides and four formsof oligosaccharides. The composition may comprise a polysaccharide orplurality of polysaccharides and five forms of oligosaccharides. Theoligosaccharides may be xylo-oligosaccharides, cello-oligosaccharides,manno-oligosaccharides, mixed-linkage glucan oligosaccharides,xyloglucan-oligosaccharides, chito-oligosaccharides,arabinoxylo-oligosaccharides, or any other suitable oligosaccharidesdescribed herein.

The composition may comprise from about 1% to 50% polysaccharides w/w,such as in the type of undigested biomass or extracted solublepolysaccharides, and about 5% to about 95% oligosaccharides w/w. Thecomposition of polysaccharides may be at least about 1%, 2%, 2.5%, 3%,4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w.Oligosaccharides in such mixtures may be present at greater than 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% w/w. The oligosaccharides may be a mixture of oneor more oligosaccharides. For instance, a composition may comprise 5%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 2.5%soluble polysaccharides and 50% oligosaccharide mixture w/w as describedelsewhere herein.

The composition may comprise about 5% polysaccharides w/w, such as inthe type of undigested biomass, and about 5% to about 95%oligosaccharides w/w. Oligosaccharides in such mixtures may be presentat greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% w/w. The oligosaccharides maybe a mixture of one or more oligosaccharides. For instance, acomposition may comprise 5% undigested biomass and 50% oligosaccharidemixture w/w as described elsewhere herein. In another instance, acomposition may comprise 5% soluble polysaccharide(s) and 50%oligosaccharide mixture w/w as described elsewhere herein.

The composition may comprise about 7% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 93%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 93% w/w of such mixtures. The oligosaccharides may be a mixtureof one or more oligosaccharides. For instance, a composition maycomprise 7% undigested biomass and 50% oligosaccharide mixture w/w asdescribed elsewhere herein. In another instance, a composition maycomprise 7% soluble polysaccharide(s) and 50% oligosaccharide mixturew/w as described elsewhere herein.

The composition may comprise about 10% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 90%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% w/w of such mixtures. The oligosaccharides may be a mixture of oneor more oligosaccharides. For instance, a composition may comprise 10%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 10%polysaccharide(s) and 50% oligosaccharide mixture w/w as describedelsewhere herein.

The composition may comprise about 12% polysaccharides w/w, such as inthe type of undigested biomass and from about 5% to about 95%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or88% w/w of such mixtures. The oligosaccharides may be a mixture of oneor more oligosaccharides. For instance, a composition may comprise 12%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 12%soluble polysaccharide(s) and 50% oligosaccharide mixture w/w asdescribed elsewhere herein.

The composition may comprise about 15% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 85%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%w/w of such mixtures. The oligosaccharides may be a mixture of one ormore oligosaccharides. For instance, a composition may comprise 15%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 15%soluble polysaccharide(s) and 50% oligosaccharide mixture w/w asdescribed elsewhere herein.

The composition may comprise about 20% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 80%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% w/wof such mixtures. The oligosaccharides may be a mixture of one or moreoligosaccharides. For instance, a composition may comprise 20%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 20%soluble polysaccharide(s) and 50% oligosaccharide mixture w/w asdescribed elsewhere herein.

The composition may comprise about 25% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 75%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% w/w ofsuch mixtures. The oligosaccharides may be a mixture of one or moreoligosaccharides. For instance, a composition may comprise 25%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 25%soluble polysaccharide(s) and 50% oligosaccharide mixture w/w asdescribed elsewhere herein.

The composition may comprise about 30% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 70%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% w/w of suchmixtures. The oligosaccharides may be a mixture of one or moreoligosaccharides. For instance, a composition may comprise 30%undigested biomass and 50% oligosaccharide mixture w/w as describedelsewhere herein. In another instance, a composition may comprise 30%soluble polysaccharide(s) and 50% oligosaccharide mixture w/w asdescribed elsewhere herein.

The composition may comprise about 40% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 60%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% w/w of such mixtures. Theoligosaccharides may be a mixture of one or more oligosaccharides. Forinstance, a composition may comprise 40% undigested biomass and 50%oligosaccharide mixture w/w as described elsewhere herein. In anotherinstance, a composition may comprise 40% soluble polysaccharide(s) and50% oligosaccharide mixture w/w as described elsewhere herein.

The composition may comprise about 50% polysaccharides w/w, such as inthe type of undigested biomass and about 5% to about 50%oligosaccharides w/w. Oligosaccharides may form at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% w/w of such mixtures. Theoligosaccharides may be a mixture of one or more oligosaccharides. Forinstance, a composition may comprise 50% undigested biomass and 50%oligosaccharide mixture w/w as described elsewhere herein. In anotherinstance, a composition may comprise 50% soluble polysaccharide(s) and50% oligosaccharide mixture w/w as described elsewhere herein.

In some embodiments, the composition or ingredient may comprise lessthan 1%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% w/w monosaccharides. Forexample, the composition may comprise less than 20% w/w monosaccharides.The composition may include from 10% to 40%, 15% to 30%, 18% to 25%, orabout 20% w/w monosaccharides. In some embodiments, the composition oringredient may comprise less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, or40% w/w glucose. For example, the composition may comprise less than 10%w/w glucose. The composition may include from 10% to 40%, 15% to 30%,18% to 25%, or about 20% w/w glucose. In some embodiments, thecomposition or ingredient may comprise less than 1%, 5%, 10%, 15%, 20%,25%, 30%, or 40% w/w xylose. For example, the composition may compriseless than 10% w/w xylose. The composition may include from 10% to 40%,15% to 30%, 18% to 25%, or about 20% w/w xylose.

In certain cases, the ratio of glucose residues to xylose residues(e.g., glucose:xylose) within the composition or ingredient may be from1:1 and 1:9, 1:1 and 1:7, 1:1 and 1:5, 1:1 and 1:3, or 1:1 and 1:2.

In certain embodiments, the composition may comprise less than 30%, 40%,50%, 60%, 65%, 70%, 75%, or 80% w/w disaccharides. For example, thecomposition may comprise less than 70% w/w disaccharides. Thecomposition may include from 10% to 95%, 15% to 90%, 20% to 80%, 30% to70%, or 40% to 60% w/w disaccharides. The composition may comprise from5% to 95%, 10% to 92.5%, 15% to 90%, 20% to 70%, 30% to 60%, or 40% to50% disaccharides. In various embodiments, the composition may compriseat least 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 15%, or 20% w/w trisaccharides.For example, the composition may comprise at least 5% w/wtrisaccharides. In various embodiments, the composition may comprise atleast 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 15%, or 20% w/w trisaccharides. Forexample, the composition may comprise at least 5% w/w trisaccharides.The composition may comprise from 1% to 75%, 2.5% to 60%, 5% to 50%, 10%to 40%, or 20% to 30% trisaccharides. In some cases, the composition maycomprise at least 0.1%, 0.5%, 1%, 2.5%, 5%, 7.5%, 10%, 15%, or 20% w/wtetrasaccharides. For example, the composition may comprise at least 1%w/w tetrasaccharides. In various cases, the composition may comprise atleast 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.5%, 1%, 2.5%, 5%, 7.5%, orw/w pentasaccharides. For example, the composition may comprise at least0.1% w/w pentasaccharides.

Use of Compositions as Ingredients

In some embodiments, the composition is an ingredient (e.g., in afoodstuff). In certain embodiments, the ingredient comprises at least50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.5% by dry weight of saccharidepresent. The ingredient may consist essentially of saccharides. Forexample, the ingredient may have less than 0.5%, 0.3%, or 0.1% by dryweight of other substances.

The ingredient may comprise an oligosaccharide mixture as describedelsewhere herein. The ingredient may comprise at least two of theoligosaccharides. For instance, it may comprise three of theoligosaccharides. It may comprise four oligosaccharides. It may comprisefive oligosaccharides. It may comprise six oligosaccharides. It maycomprise seven oligosaccharides.

In some embodiments, the ingredient comprises cello-oligosaccharides,for instance, cello-oligosaccharides in combination withxylo-oligosaccharides. An alternative ingredient may comprisecello-oligosaccharides in combination with manno-oligosaccharides.

Ingredients may be used to prepare finished products. The ingredient mayalso be treated in some physical or chemical way before or duringincorporation into a foodstuff, cosmetic, or nutraceutical. It may bedirectly incorporated into a product, or it may be incorporated into,for example, a dough, cake mixture, chocolate mixture, or otherfoodstuff precursor; a cosmetic base composition; or a nutraceutical,and, for example, be cooked or otherwise treated in a way which maycause chemical modification, a change of texture, a change of color, orother modification.

A foodstuff, cosmetic, or nutraceutical may be produced from aningredient described herein. For example, in the food industry, thesaccharide formulations produced by the current method may be used assweeteners, bulking agents, added dietary fiber, or humectants. Theingredient may be used as a sugar substitute. The ingredient may beincorporated into cakes, breads, or other baked goods, or into chocolateor other confectionery such as toffee, fudge, meringue, jam, jelly, orcaramel; or drinks, for example, to provide favorable taste or colorcharacteristics or to increase dietary fiber content. In certaininstances, the ingredient may be incorporated into animal feed, forexample, either as an isolated ingredient or by utilizing the enzymaticreaction mixture directly as feed.

In the cosmetics industry, saccharides can be useful as ingredients, asthey may improve texture and moisture retention, act as UV-absorbingmolecules, maintain a gel or cream structure, and/or serve as bulkingagents. The compositions described herein can be incorporated intonutraceutical compositions, as the dietary fiber they provide canencourage digestive health, well-regulated gut flora, and other benefitsto wellbeing. In this context, they may also function as an ingredientin a probiotic drink or other prebiotic or probiotic formulation.

Compositions or ingredients as described herein may be used to alter oneor more properties of the finished product. Such properties include, butare not limited to, sweetness, texture, mouthfeel, binding, glazing,smoothness, moistness, viscosity, color, hygroscopicity, flavor,bulking, water-retention, caramelization, surface texture,crystallization, structural properties, and dissolution.

In some cases, the compositions and/or ingredients described herein mayprovide a property to a finished product which is comparable to orbetter than the same property as provided by a saccharide mixturecomprising primarily monosaccharides and/or disaccharides. The controlcomposition may be a saccharide used commonly in consumables, forinstance, a monosaccharide composition such as glucose, fructose, etc, adisaccharide composition such as sucrose or an artificial sugarcomposition. The control composition may be table sugar, corn syrup,high-fructose corn syrup, or any other suitable composition. The term“comparable,” as used herein, generally means that the two compositionsmay be up to 100%, up to 95%, up to 90%, or up to 80% identical. Forinstance, comparable can mean that the composition is up to 90%identical to the control composition.

In some cases, the compositions described herein may be used assweetener compositions. Sweetener compositions may be used by themselvesor as an ingredient in a finished product. The compositions describedherein may provide about the same level of sweetness or greatersweetness than an identical amount of a control composition wherein thecontrol composition comprises primarily monosaccharides and/ordisaccharides. The compositions described herein may be used to replacethe control composition as the sweetener in a finished product. In somecases, the sweetness of a composition may be 5%, 10%, 15%, 20%, 30%,40%, 50%, 70%, 80%, 90%, or 100% more than an identical amount of thecontrol composition.

The compositions described herein may provide a comparable flavorprofile or better flavor profile than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a flavor enhancer in afinished product. In some cases, the flavor of a composition may be 5%,10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, or 100% more than anidentical amount of the control composition.

The compositions described herein may provide a comparable textureprofile or better texture profile than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a texture enhancer ina finished product.

The compositions described herein may provide a comparable bindingprofile or better binding profile than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a binding enhancer ina finished product.

The compositions described herein may provide a comparable glazingprofile or better glazing profile than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a glazing enhancer ina finished product.

The compositions described herein may provide a comparable moistness orbetter moistness than an identical amount of a control compositionwherein the control composition comprises primarily monosaccharidesand/or disaccharides. The compositions described herein may be used toreplace the control composition to provide moistness in a finishedproduct.

The compositions described herein may provide a comparable color profileor better color profile than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a color enhancer in afinished product.

The compositions described herein may provide a comparable dissolutionprofile or better dissolution profile than an identical amount of acontrol composition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. The compositions described hereinmay be used to replace the control composition as a dissolution enhancerin a finished product. In some cases, the dissolution of a compositionmay be 5%, 10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, or 100% morethan an identical amount of the control composition.

The compositions described herein may provide a comparable mouthfeel orbetter mouthfeel than an identical amount of a control compositionwherein the control composition comprises primarily monosaccharidesand/or disaccharides.

The compositions described herein may provide a comparable viscosity orbetter viscosity than an identical amount of a control compositionwherein the control composition comprises primarily monosaccharidesand/or disaccharides.

The compositions described herein may provide a comparablehygroscopicity or better hygroscopicity than an identical amount of acontrol composition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. In some cases, the hygroscopicityof a composition may be 5%, 10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%,or 100% more than an identical amount of the control composition.

The compositions described herein may provide a comparablewater-retention or better water-retention than an identical amount of acontrol composition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. In some cases, the water-retentionof a composition may be 5%, 10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%,or 100% more than an identical amount of the control composition.

The compositions described herein may provide a lower caloriecomposition than an identical amount of a control composition whereinthe control composition comprises primarily monosaccharides and/ordisaccharides. In some cases, the calorie count of a composition may be5%, 10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, or 100% less than anidentical amount of the control composition.

The compositions described herein may provide a lower glycemic indexthan an identical amount of a control composition wherein the controlcomposition comprises primarily monosaccharides and/or disaccharides. Insome cases, the glycemic index of a composition may be 5%, 10%, 15%,20%, 30%, 40%, 50%, 70%, 80%, 90%, or 100% less than an identical amountof the control composition.

The compositions described herein may provide a comparable bulking orbetter bulking than an identical amount of a control composition whereinthe control composition comprises primarily monosaccharides and/ordisaccharides.

The compositions described herein may provide a comparablecaramelization or better caramelization than an identical amount of acontrol composition wherein the control composition comprises primarilymonosaccharides and/or disaccharides.

The compositions described herein may provide a comparable surfacetexture or better surface texture than an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides.

The compositions described herein may provide a comparablecrystallization or better crystallization than an identical amount of acontrol composition wherein the control composition comprises primarilymonosaccharides and/or disaccharides.

The compositions described herein may provide comparable structuralproperties as an identical amount of a control composition wherein thecontrol composition comprises primarily monosaccharides and/ordisaccharides.

The compositions described herein may provide less aftertaste comparedto an identical amount of a control composition wherein the controlcomposition comprises primarily monosaccharides and/or disaccharides.

Different compositions of oligosaccharides may have improved dissolutionprofiles, hygroscopicity profiles, and taste profiles compared to theoligosaccharides used alone.

The compositions or ingredients as described herein may be used toincrease the fiber content of a finished product such as a foodstuff ora nutraceutical. The compositions may provide a higher level of fiber inthe finished product as compared to an identical amount of a controlcomposition wherein the control composition comprises primarilymonosaccharides and/or disaccharides. In some cases, the compositionsmay improve the fiber content of the finished product withoutnegatively, or substantially negatively, affecting any other propertiessuch as taste, sweetness, mouthfeel, texture, binding, or any otherproperties described herein. In some cases, the fiber content of acomposition may be 5%, 10%, 15%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, or100% more than an identical amount of the control composition.

Ingredients may be used to alter the properties of a finished productsuch as foodstuff or nutraceutical or cosmetic. In order to alter theproperties of the finished products, the finished products mayadditionally comprise a polysaccharide, for example, a cellulosicpolysaccharide, such as cellulose, or a polysaccharide derivative, forexample, a cellulose derivative, such as carboxymethylcellulose, or apolysaccharide aggregate, for example, a portion of lignocellulosicbiomass. In some instances, the finished products can comprise fromgreater than 0% to 40% by dry weight of polysaccharide, polysaccharidederivative, or polysaccharide aggregate, for example, from greater than1% to 30% by dry weight of polysaccharide, polysaccharide derivative, orpolysaccharide aggregate, for example, from greater than 5% to 25% bydry weight of polysaccharide, polysaccharide derivative, orpolysaccharide aggregate, for example, from greater than 10% to 20% bydry weight of polysaccharide, polysaccharide derivative, orpolysaccharide aggregate.

The concentration of a composition comprising polysaccharides and amixture of oligosaccharides in a finished product may be anywhere from0.1% to 40% w/w. The concentration of a composition comprisingpolysaccharides and a mixture of oligosaccharides in a finished productmay be from about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1%to about 5%, about 0.1% to about 10%, about 0.1% to about 15%, about0.1% to about 20%, about 0.1% to about 25%, about 0.1% to about 30%,about 0.1% to about 35%, about 0.1% to about 40%, about 0.5% to about1%, about 0.5% to about 5%, about 0.5% to about 10%, about 0.5% to about15%, about 0.5% to about 20%, about 0.5% to about 25%, about 0.5% toabout 30%, about 0.5% to about 35%, about 0.5% to about 40%, about 1% toabout 5%, about 1% to about 10%, about 1% to about 15%, about 1% toabout 20%, about 1% to about 25%, about 1% to about 30%, about 1% toabout 35%, about 1% to about 40%, about 5% to about 10%, about 5% toabout 15%, about 5% to about 20%, about 5% to about 25%, about 5% toabout 30%, about 5% to about 35%, about 5% to about 40%, about 10% toabout 15%, about 10% to about 20%, about 10% to about 25%, about 10% toabout 30%, about 10% to about 35%, about 10% to about 40%, about 15% toabout 20%, about 15% to about 25%, about 15% to about 30%, about 15% toabout 35%, about 15% to about 40%, about 20% to about 25%, about 20% toabout 30%, about 20% to about 35%, about 20% to about 40%, about 25% toabout 30%, about 25% to about 35%, about 25% to about 40%, about 30% toabout 35%, about 30% to about 40%, or about 35% to about 40% w/w. Theconcentration of a composition comprising polysaccharides and a mixtureof oligosaccharides in a finished product may be about 0.1%, about 0.5%,about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, or about 40% w/w. The concentration of a compositioncomprising polysaccharides and a mixture of oligosaccharides in afinished product may be at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%,30%, or 35% w/w. The concentration of a composition comprisingpolysaccharides and a mixture of oligosaccharides in a finished productmay be at most 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% w/w.

In some cases, the oligosaccharide mixtures (e.g.,cello-oligosaccharides and xylo-oligosaccharides) may form at least 20%,30%, 40%, 50%, 60%, or 70% w/w of the consumable composition oringredient. For example, 50% w/w of a combination ofcello-oligosaccharides and xylo-oligosaccharides may form the consumablecomposition or ingredient.

In some instances, the ingredient may include a maize cob extract (MCE).The MCE may be a mixture of oligosaccharides composed primarily ofarabinoxylo-oligosaccharides, xylo-oligosaccharides,cello-oligosaccharides, and cellulose. In certain instances, theoligosaccharides may be non-digestible or substantially non-digestible.The arabinoxylo-oligosaccharides can be oligomers with xylose backboneslinked by β-(1→4)-bonds substituted with arabinose side-chains. Thearabinoxylo-oligosaccharides may be non-digestible. Thearabinoxylo-oligosaccharides can be produced by hydrolysis ofarabinoxylan (polysaccharide of β-(1→4)-bonded xylose units substitutedwith arabinose side-chains). Furthermore, thearabinoxylo-oligosaccharides may have a degree of polymerization (DP) of3 to 15.

In various instances, the xylo-oligosaccharides may be oligomers withxylose backbones linked by β-(1→4)-bonds. The xylo-oligosaccharides maybe non-digestible. The xylo-oligosaccharides may be produced byhydrolysis of arabinoxylan. Moreover, the xylo-oligosaccharides may havea DP of 2 to 8.

In certain instances, the cello-oligosaccharides may be oligomers withglucose backbones linked by β-(1→4)-bonds. The cello-oligosaccharidesmay be non-digestible. The cello-oligosaccharides may be produced byhydrolysis of cellulose (polysaccharide of β-(1→4)-bonded glucoseunits). Furthermore, the cello-oligosaccharides may have a DP of 2 to 4,with the majority having a DP of 2.

IV. Exemplary Embodiments

Exemplary Method of Extracting Soluble Polysaccharides for SubsequentCombination with Generated Oligosaccharides

FIG. 8 is a simplified flow diagram showing an embodiment of a method ofextracting soluble polysaccharides for subsequent combination withgenerated oligosaccharides to form an ingredient.

In the illustrated method, the extraction or removal of at least aportion of the soluble polysaccharides 140 before an enzyme treatment150 can ensure that at least a portion of the soluble polysaccharidesare retained or saved for combination 144 with the generatedoligosaccharides to form the ingredient 105. In some other methods (notshown), the soluble polysaccharides can be digested by the one or moreenzymes because the soluble polysaccharides are not extracted prior tothe enzyme treatment. The ingredient 105 of the illustrated method canbe a sweetener or sugar substitute that can remain substantially solubleor entirely soluble. Accordingly, the ingredient 105 can be delivered asa syrup-like product (e.g., a viscous liquid). In some cases, theingredient 105 can be a replacement or partial replacement for cornsyrup, high-fructose corn syrup, maple syrup, honey, treacle, goldensyrup, molasses, dextrose syrup, fructose syrup, agave nectar, datesyrup, brown rice syrup, coconut syrup, corn syrup or other suitableliquid sweeteners in a foodstuff.

As depicted, biomass 107 (e.g., corncob or any other suitable biomass)can be physically pretreated 110 (e.g., by chipping or any othersuitable method of physically pretreating a biomass). The physicallytreated biomass 112 can then be subjected to or undergo a thermochemicalpretreatment 130 (e.g., 15% w/v chipped corncob can be heated in 1% w/vNaOH for one hour). In some embodiments, the thermochemical pretreatment130 can be followed by a neutralization step (not shown) beforeextraction of the soluble compounds or material 140 from the physicallypretreated biomass 112. Extraction of the soluble compounds or material140 can include removing a liquid portion (e.g., a supernatant) of thephysically pretreated biomass 112. The liquid portion can includesoluble compounds 146 from the physically pretreated biomass 112. Thesoluble compounds 146 can include soluble polysaccharides. In certainembodiments, 15% of the liquid portion (including the soluble compoundsor material 146) can then be extracted or removed from thethermochemically pretreated biomass 112. The extracted portion includingthe soluble compounds or material 146 (e.g., the solublepolysaccharides) can then be subjected to one or more purification steps142 b (e.g., ultrafiltration) to enrich the soluble polysaccharides 147.

Furthermore, the liquid portion that is not extracted at step 140 caninclude soluble compounds or material 146 and insoluble compounds ormaterial 148. For example, in the case where 15% of the liquid portionis extracted as described above, the remaining portion of the physicallypretreated biomass 112 (including 85% of the liquid portion) can includesoluble polysaccharides and insoluble polysaccharides. The solutionincluding the soluble compounds or material 146 and insoluble compoundsor material 148 can then be subjected to or undergo an enzyme treatment150, as disclosed herein. For example, one or morepolysaccharide-cleaving enzymes can be added to the solution includingthe soluble compounds or material 146 and insoluble compounds ormaterial 148 to 0.5% w/v and incubated at 50° C. for 24 hours. Theenzyme-treated biomass 151 can then be treated (e.g., filtered) 152 toremove at least a portion of the undigested biomass. In certain cases,the removed undigested biomass can be disposed of or rejected. Thedigested biomass 141 can then be subjected to or undergo purification142 a (e.g., ion-exchange chromatography, nanofiltration,microfiltration, ultrafiltration, or any other suitable method ofpurification) to enrich for oligosaccharides 154 as described herein.The extracted, isolated, and/or purified oligosaccharides 154 and theextracted, isolated, and/or purified soluble polysaccharides 147 can becombined, mixed, and or spray dried to form the ingredient 105.

Exemplary Method of Pretreating Biomass to Remove Monosaccharides and/orDisaccharides

FIG. 9 is a simplified flow diagram showing an embodiment of a method ofpretreating biomass to remove monosaccharides and/or disaccharides priorto an enzyme treatment.

The embodiment of FIG. 9 may include components or steps that resemblethe components or steps of the embodiment of FIG. 8 in some respects.For example, the embodiment of FIG. 9 includes the step of physicallypretreatment 110 that may resemble the physical pretreatment 210 of FIG.8. It will be appreciated that the illustrated embodiments may haveanalogous features. Accordingly, like features are designated with likereference numerals, with leading digits added to increment eachreference numeral by 100. For instance, the physical pretreatment isdesignated “110” in FIG. 8 and an analogous physical pretreatment isdesignated as “210” in FIG. 9. Relevant disclosure set forth aboveregarding similarly identified features thus may not be repeatedhereafter. Moreover, specific features of the method and relatedcomponents or steps shown in FIG. 9 may not be shown or identified by areference numeral in the drawings or specifically discussed in thewritten description that follows. However, such features may clearly bethe same, or substantially the same, as features depicted in otherembodiments and/or described with respect to such embodiments.Accordingly, the relevant descriptions of such features apply equally tothe features of the method and related components or steps of FIG. 9.Any suitable combination of the features, and variations of the same,described with respect to the method illustrated in FIG. 8, can beemployed with the method and components or steps of FIG. 9, and viceversa. This pattern of disclosure applies equally to further embodimentsdepicted in subsequent figures and/or described hereafter.

As illustrated, a gentle pretreatment 220 (e.g., a washing or incubationcycle as provided herein) of a physically pretreated biomass 212 caninclude removing 224 the soluble compounds 246. In certain cases, thesoluble compounds 246 can include monosaccharides and/or disaccharides.The removed soluble monosaccharides and/or disaccharides can then bediscarding and/or rejected. Accordingly, the gentle pretreatment 220 canbe conducted or performed to remove soluble monosaccharides and/ordisaccharides from the biomass 207.

Biomass 207 can be physically pretreated 210 (e.g., chipped) and thenthe physically pretreated biomass 212 can be subjected to or undergo agentle pretreatment 220, for example, washed or incubated (e.g., inwater at 25° C. for 30 minutes). Soluble saccharides 246 (e.g., solublemonosaccharides and/or disaccharides) can then be removed from thesolution including the gently pretreated biomass 226. The gentlypretreated biomass 226 can then be subjected to or undergo a strongpretreatment 230. In certain cases, the strong pretreatment 230 can be athermochemical pretreatment. For example, the gently pretreated biomass226 can be treated in 1% w/v NaOH at 100° C. for 60 minutes. In someembodiments, the strong pretreatment 230 can be followed by aneutralization step (not shown) before the enzyme treatment 250. Thestrongly pretreated biomass 232 can then be treated with enzymes 250 asdiscussed herein. Furthermore, downstream processing 260 can then beconducted on the enzyme-treated biomass 251 to generate the ingredient205.

Additional Exemplary Embodiments of Extracting Soluble Polysaccharidesfor Combination with Oligosaccharides

In some instances, the disclosure relates to novel methods of treatingplant biomass materials for the production of a foodstuff, cosmetic, ornutraceutical ingredient.

Sugary foods and drinks are an important part of culture and lifestylehabits across the world, but the sugar they contain has been linked toobesity, diabetes, poor dental health, and disruptive behavior inpeople. Because of this, consumer preferences have been shifting awayfrom sugar-containing foods, and governments are increasinglyimplementing regulation to encourage the consumption of less sugar.

As such, industry has been searching for suitable low-calorie sweetenersfor many decades to substitute for sugar in food and beverages.Unfortunately, many sugar substitutes are produced from non-naturalresources, and often offer bitter undertones or other unpleasant tastesalong with their sweetness, both of which consumers find unappealing.Moreover, while many sweeteners are able to mimic the sweetness of sugarin food and drinks, few are able to mimic the broad range of roles thatsugar plays in food, such as adding bulk, modulating texture, providingstructure, acting as a preservative, and modulating color and flavorthrough caramelization and Maillard reactions.

Dietary fiber is an important part of a positive diet, and helpsmaintain digestive health and a well-regulated gut flora. Such fibercomprises saccharides of varying chain lengths and types. In addition tobeing found naturally in a wide spectrum of foods, fiber can also beproduced separately and added to other foods during their manufacture.

Biomass is a good source of saccharides that can be used to replacesugar and add fiber to food products. However, there remains a need tooptimize the process by which these saccharides are obtained from thebiomass and processed into the compositions useful as a foodstuff,cosmetic, or nutraceutical ingredient.

Firstly, the saccharides generally need to be produced by controlledbreakdown. Different amounts of different sized saccharides in theingredient can affect its nutritional values, and also other propertiessuch as hygroscopicity which in turn affect properties such as thetexture of the product the ingredient is used to make. It can also bedesirable for the ingredient to comprise polysaccharide aspolysaccharide can improve gastrointestinal tolerance. However, due tothe speed at which some particularly desirable polysaccharides breakdown during the enzymatic reaction of previously known methods, it isdifficult to isolate and then incorporate into the ingredient thesedesirable polysaccharides.

Furthermore, food products that require a smooth texture, such as candy,chocolate, and yoghurt, generally require the ingredient to be solubleto achieve the smooth texture. Ingredients comprising insolublepolymeric material can result in a gritty texture. However, due to theinsolubility of certain polysaccharides, it can be difficult to make acomposition comprising polysaccharides that is entirely soluble,particularly in a one-pot process from a single piece of biomass.Typically, soluble polysaccharides break down quicker than insolublepolysaccharides so after exposing a plant biomass to enzymes, like ofpreviously known methods, the soluble polysaccharides are entirelybroken down and only insoluble polysaccharides are left.

Surprisingly, methods have been identified here that can allowpolysaccharides to be isolated and incorporated into a solublefoodstuff, cosmetic, or nutraceutical ingredient comprisingoligosaccharides, which thus maintains the benefit of increasing thegastrointestinal tolerance of the ingredient as well as allowing it tobe used in food products with a smooth texture. The polysaccharides canbe isolated from the same plant biomass as that which the other desiredsaccharides are obtained from providing an efficient and stream-linedproduction process.

Accordingly, in a first aspect of the disclosure there is provided amethod for producing a foodstuff, cosmetic, or nutraceutical ingredientcomprising the steps of:

-   -   a) providing a plant biomass comprising one or more soluble        polysaccharides and one or more insoluble polysaccharides;    -   b) treating the plant biomass to dissolve the one or more        soluble polysaccharides;    -   c) removing a portion of the dissolved one or more soluble        polysaccharides;    -   d) reacting the remaining plant biomass with one or more enzymes        to form one or more oligosaccharides;    -   e) removing the one or more oligosaccharides; and    -   f) combining the portion of the dissolved one or more soluble        polysaccharides from step (c) and the one or more        oligosaccharides from step (e) to form the ingredient.

As such, there is also provided a foodstuff, cosmetic, or nutraceuticalingredient obtainable by the methods of the disclosure.

In another aspect of the disclosure there is provided a foodstuff,cosmetic, or nutraceutical liquid ingredient comprising at least oneoligosaccharide selected from the list consisting of:

-   -   i) cello-oligosaccharide having a degree of polymerization of        from two to six;    -   ii) xylo-oligosaccharide having a degree of polymerization of        from two to twelve;    -   iii) manno-oligosaccharide having a degree of polymerization of        from two to twelve;    -   iv) mixed-linkage glucan oligosaccharide having a degree of        polymerization of from two to five;    -   v) xyloglucan oligosaccharide having a degree of polymerization        of from four to twelve; and    -   vi) chito-oligosaccharide having a degree of polymerization of        from two to twelve; and at least one polysaccharide selected        from the list consisting of:    -   i) xylan;    -   ii) mannan;    -   iii) cellulose derivative;    -   iv) mixed-linkage glucan;    -   v) xyloglucan; and    -   vi) chitosan;

wherein the liquid ingredient comprises at least 20% by dry weight ofthe at least one oligosaccharide and at least 2% by dry weight of the atleast one polysaccharide and wherein the liquid ingredient has aviscosity of from 5 to 100,000 cps.

Preparing the foodstuff, cosmetic, or nutraceutical ingredient in themanner provided herein can allow efficient use of biomass byincorporating oligomeric and polymeric material from the same biomasssource to make a soluble ingredient. Furthermore, the methods can allowfor purification, derivatization, or other modification, as well ascontrol of oligomeric and polymeric proportions, which can improve thefunctional properties, nutritional properties, and tolerance of theingredient.

Any substance which comprises suitable polysaccharides may be the plantbiomass. As the foodstuff, cosmetic, and nutraceutical industries use abroad variety of oligosaccharides, the polysaccharides suitable in themethod are not particularly limited. Plant biomass suitable forproducing the oligosaccharide profile of the current disclosure maycomprise, for example, cellulose, lignocellulose, chitin, chitosan,xylan (such as glucuronoxylan, arabinoxylan, and glucuronoarabinoxylan),xyloglucan, mixed-linkage glucan, and/or mannan (such as glucomannan,galactomannan, or galactoglucomannan), however, any plant biomass whichcan be suitably acted upon is envisaged. The one or more solublepolysaccharides that the plant biomass comprises may include any one ofthe following: mannans, mixed-linkage glucans, lignocellulose,hemicellulose, certain cellulose derivatives such as cellulose acetate,hydroxyethylcellulose, and hydroxymethylcellulose, and chitosan. In someembodiments, the plant biomass comprises hemicellulose. In certainembodiments, the hemicellulose comprises xylan and/or mannan.

As such, the plant biomass may be grain, grain chaff, bean pods, seedcoats, and/or other seed materials; seaweeds; corn stover, straw,bagasse, miscanthus, sorghum residue, switch grass, bamboo, and/or othermonocotyledonous tissue; water hyacinth, leaf tissue, roots, and/orother vegetative matter; and/or any combination of suitable plantbiomasses. In some cases, the plant biomass comprises, or suitablyconsists of, sugar cane biomass (such as sugar cane bagasse), cornbiomass (such as corncob or corn stover), wheat biomass (such as wheatstraw or wheat bran), hardwood or softwood. In certain cases, the plantbiomass comprises corncob, sugar cane bagasse, wheat straw, or ricestraw.

In various instances, in step (b) the “treating” is a thermochemicaltreatment of the plant biomass. “Thermochemical,” as used herein,generally refers to heating above room temperature (room temperature canbe about 20° C. to 22° C.) the plant biomass in a chemical substance,such as heating in a solution comprising water, alkali, or ionicsolvents. The thermochemical step can physically and chemically modifychemical components of the plant biomass. For example, the freehydroxide ions from the water or alkali can disrupt hydrogen bondsbetween saccharides enabling the solubilization of some types ofsaccharides, for example, hemicelluloses, and can better enable theenzyme in the subsequent step to more easily break up the saccharides.These disrupted hydrogen bonds may be between monomers of the samesaccharide chain which contribute to the chain's tertiary structure. Thedisrupted hydrogen bonds may also be between monomers of differentsaccharide chains which contribute to the quaternary structure of morethan one chain. Subsequently, the treatment can result in the one ormore polysaccharides that are soluble (i.e., the polysaccharides thatare particularly susceptible to the disruption of the hydroxide ions,especially, for example, hemicelluloses) to be dissolved into thechemical substance used. The one or more polysaccharides that areinsoluble, such as cellulose, do not dissolve into the chemicalsubstance.

The heating of the treatment step (b) may be at a range of temperatures,suitably of from 30° C. to 180° C., 50° C. to 150° C., or from 70° C. to120° C. Higher temperatures can help the soluble polysaccharides todissolve quicker, however, temperatures that are too high can be moredifficult to achieve in an efficient and cost-effective manner, and canchemically modify the biomass components, including the saccharides(e.g., in an undesirable manner).

The heating may occur for a range of time scales, particularly largeamounts of biomass may be exposed to heating for a longer period oftime, which can be adjusted accordingly. For example, the heating of theplant biomass can be of from 1 minute to 72 hours, 10 minutes to 24hours, 20 minutes to 12 hours, or 25 minutes to 8 hours.

In some instances, the thermochemical treatment may comprise heating theplant biomass in water, i.e., at a neutral pH of about pH 7.

In certain instances, the thermochemical treatment may comprise heatingthe plant biomass in an alkali solution having a pH of from 8 to 14, 9to 14, or 10 to 14. The solution may comprise, or suitably consist of,any one of the alkalis selected from: sodium hydroxide, potassiumhydroxide, sodium carbonate, calcium carbonate, calcium hydroxide,ammonium sulfate, ammonium hydroxide, and aqueous ammonia. In variousinstances, the alkali may be sodium hydroxide. A combination of thelisted alkalis is also envisaged.

Multiple different sequential thermochemical treatment steps are alsoenvisaged. For example, there may be two sequential thermochemicaltreatments, there may be three sequential thermochemical treatments, orthere may be four or more sequential thermochemical treatments. In somecases, the biomass may be thermochemically treated in a neutral aqueoussolution and then thermochemically treated in an alkaline aqueoussolution.

After the treating step, step (c) can comprise removing a portion of thedissolved one or more soluble polysaccharides. The purpose of this stepcan be to isolate and remove the soluble polysaccharides from the plantbiomass so that they do not get broken down and therefore lost in thesubsequent enzymatic reaction. This can enable the use of thesepolysaccharides when forming the ingredient in a later step. All of thedissolved polysaccharides or a portion of them may be removed dependenton the desired amount in the final ingredient. The solublepolysaccharides can be removed using simple steps such as filtering thechemical substance, of which the soluble polysaccharides are dissolvedin.

Step d) comprises reacting the remaining plant biomass, which may be inthe form of a solution and/or a suspension, with one or more enzymes toform the one or more oligosaccharides. The soluble and insolublepolysaccharides present in the remaining plant biomass solution and/orsuspension can be partially or fully cleaved by the one or more enzymesinto oligosaccharides (e.g., useful oligosaccharides), potentiallyleaving partially cleaved, or uncleaved, polysaccharides, which mayinclude cellulose, xylan (such as glucuronoxylan, arabinoxylan, orglucuronoarabinoxylan), mannan (such as glucomannan, galactomannan, orgalactoglucomannan), mixed-linkage glucan, xyloglucan, chitin, chitosan,or lignocellulose.

The enzyme reaction may take place in solution and/or suspension, in asuitable reaction vessel. The enzyme reaction may take place at atemperature or temperature protocol suitable for the particularcombination of enzyme and plant biomass, the reaction may be allowed toprogress for a certain amount of time, until the products have reached adesired concentration, or until some other requirement has been met.

In order to ensure optimal contact between the enzymes and the plantbiomass, the reaction mixture may be agitated, either constantly or atintervals. The agitation may take the form of rhythmically moving theentire reaction vessel, of a fan or other stirring device, of a bubblesparging, or any other method of agitation.

The enzymatic reaction may be a microbial fermentation. The temperatureand reaction time can be suitable for the growth of the microbialorganism used. The microbial organism may be genetically altered toproduce an enzyme suitable for the production of an oligosaccharide ofthe present disclosure. The microbe may be, for example, a bacterium,for example, Escherichia coli, or a fungus, such as Saccharomycescerevisiae, Aspergillus niger, or Trichoderma reesei.

Further embodied in the present disclosure is an expression vectorsuitable for modifying the subject microorganism such that it producesan enzyme or mixture of enzymes of the current disclosure. Wheredesired, the expression vector, which may be a plasmid or any othernucleic acid able to induce production of the enzyme, may comprise oneor more of the following regulatory sequences so as to control theexpression of the exogenous enzyme: regulatory sequences of a heat shockgene, regulatory sequences of a toxicity gene, and regulatory sequencesof a spore formation gene.

The enzymatic reaction can be carried out at a temperature ortemperature protocol suitable to the enzymes and substrates used. Forexample, it may be carried out at a constant temperature in the range offrom about 10° C. to about 100° C., about 20° C. to about 70° C., orabout 30° C. to about 60° C. If the enzymatic reaction takes the form ofa microbial fermentation the temperature may be suitable for such, forexample, the enzymatic reaction may comprise the growth of E. coliand/or the temperature may be constant and about 37° C.

The pH of the solution or suspension may affect the activity of theenzymes. Control of pH may assure that an enzymatic reaction proceeds ata suitable rate. The enzymatic reaction of the present disclosure maytake place at a pH in the range of from about 2 to about 10, about 3 toabout 8, or about 4 to about 6.

The enzymatic reaction can be allowed to continue for a certain timeperiod before being quenched, and the products isolated or otherwisecollected. This time period may be from about 1 minute to about 6 days,from about 0.5 days to about 5 days, or from about 16 hours to about 96hours. The reaction may alternatively be allowed to proceed until nofurther catalysis occurs.

The enzymatic reaction can be allowed to continue to run until there isless than 75% undigested polysaccharide-containing plant biomassesremaining, less than 70%, less than 65%, less than 55%, or less than50%. This can be monitored or checked by reducing end assays, such asthe anthrone assay and/or by chromatographic methods such as thin-layerchromatography and high-performance anion exchange chromatography. Thereaction may run until all polysaccharides are converted tooligosaccharides.

There are many enzymes that are suitable for use in the enzymaticreaction of the present method. For example, “lytic polysaccharidemonooxygenase” and “LPMO,” which are a class of enzymes able tooxidatively cleave polysaccharides using a copper comprising moiety andusing an oxygen source, such as a molecule of dioxygen, peroxide, or anyother oxygen source; and a suitable reducing agent. As such, when anLPMO is used, the enzymatic reaction may be carried out under aerobicconditions. Suitable reducing agents are not particularly limited, butexamples include ascorbic acid, gallic acid, cysteine, NADH, NADPH,pyrogallol, dithiothreitol, cyanoborohydrides, borohydrides,photosynthetic pigments, lignin, lignols, and a combination ofcellobiose and cellobiose dehydrogenase. A wide variety ofphotosynthetic pigments may be used. In some embodiments, thylakoids andpurified fractions, or chlorophyllin may be used, and light may besupplied. LPMOs can be selected from the following families: AA9, AA10,AA11, AA13, AA14, and AA15. In various cases, the LPMO may be PaLPMO9E(SEQ ID NO:1), an AA9 LPMO originally isolated from the ascomycetefungus Podospora anserina or the LPMO may be an AA9 LPMO fromTrichoderma reesei (SEQ ID NO:23).

Aerobic conditions may comprise the addition of oxygen, which may beprovided by aeration of the substrate mixture with an oxygen-comprisinggas, such as air. Aeration may be conducted by the introduction ofoxygen-comprising air bubbles into the aqueous substrate mixtures byvarious systems, such as an air-injector, an aeration frit, a membranesystem, or an internal-loop airlift reactor. The concentration ofmolecular oxygen in the enzymatic reaction may be from about 4 mg/L toabout 14 mg/L.

Another type of enzyme that can be used in the method is a “cellulase,”which has hydrolytic activity against cellulose, for example,endo-1,4-beta-glucanase, cellobiohydrolase, and/or beta-glucosidaseactivities. Such enzymes are able to cleave glycosidic bonds in one ormore forms of cellulose, including cellulose found in plant biomass. Indoing so, they produce products including glucose andcello-oligosaccharides. In certain cases, the beta-glucanases mayinclude enzymes from GH5, GH7, and GH12 enzyme, such as those derivedfrom Aspergillus niger (SEQ ID NO:12, 13 and 14) and Trichoderma reesei(SEQ ID NOs:24 and 25).

Another type of enzyme is “cellobiohydrolase,” which has hydrolyticactivity against cellulose and produces mainly cellobiose as a product.Cellobiose is a disaccharide and is a cello-oligosaccharide. Suchenzymes are able to cleave glycosidic bonds in one or more forms ofcellulose, including cellulose found in plant biomass. In variousinstances, cellobiohydrolases may be from GH6 and GH7 enzyme families,Cel6A or Cel7A enzymes derived from Trichoderma reesei (SEQ ID NOs:10and 11, respectively).

Another type of enzyme is “beta-glucosidase,” which has hydrolyticactivity against cellulose and produces mainly glucose as a product.Such enzymes are able to cleave glycosidic bonds in one or more forms ofcellulose, including cellulose found in plant biomass. In someembodiments, beta-glucosidases may include GH3 beta-glucosidases, suchas one from Trichoderma reesei (SEQ ID NO: 22).

Another type of enzyme is a lichenase, which may be selected from: theGH5, GH7, GH8, GH9, GH12, GH16, GH17, or GH26 families. In someembodiments, the lichenase may be a GH16 enzyme, for example, a GH16enzyme derived from Bacillus subtilis (SEQ ID NO:2). The enzyme is ableto act on, for example, mixed-linkage glucans, which are glucanscomprising a mixture of β-1,3 and β-1,4 linkages, and may cleave them atβ-1,4 glycosidic bonds. In the case in which the lichenase acts on amixed-linkage glucan, the β-glucans produced may fall largely within thesize range of from about 3 to about 7 residues, so they are particularlyuseful in the food, cosmetics, and nutraceutical industries.Mixed-linkage glucans are abundant in members of the grass and horsetailfamilies, and as such, grass-based biomasses such as straw have highlevels of mixed-linkage glucans, and may be acted upon usefully withlichenases.

Another type of enzyme is a xylanase, which may act on, for example,plant biomass comprising a xylan backbone. The xylanase may be, forexample, a glucuronoxylanase, an arabinoxylanase, or aglucuronoarabinoxylanase. The enzyme may be active on a variety ofpolymers having a xylan backbone, such as glucuronoxylan, arabinoxylan,and glucuronoarabinoxylan. These polymers are abundant in various plantbiomass, for example, both hardwood and softwood may comprise suitablepolysaccharides, with hardwood often comprising glucuronoxylan andsoftwood often arabinoglucuronoxylan. In some instances, xylanases mayinclude GH5 xylanases from Ruminiclostridium thermocellum (SEQ ID NO:3)and Gonapodya prolifera (SEQ ID NO:4), and GH30 xylanases from Dickeyachrysanthemi (SEQ ID NO:5), Bacillus subtilis (SEQ ID NO:6), Bacteroidesovatus (SEQ ID NO:7), and Trichoderma reesei (SEQ ID NO: 15).

Other enzymes useful in the disclosure may include xyloglucanases andxyloglucan endoglucanases (XEGs), which are produced by numerousorganisms, including plant-pathogenic microbes. They are able to act onxyloglucan, a hemicellulosic β-1,4 glucan chain abundant in the primarycell wall of higher plants, which is decorated with xylose, some of thexylose residues being further decorated with other residues, such asgalactose. When suitable xyloglucanases or XEGs act on xyloglucan, theproducts comprise xyloglucan oligosaccharides having a main chain of alength useful in the foodstuff, cosmetics, and nutraceutical industries.In some cases, xyloglucanases may include a GH5 xyloglucanase fromBacteroides ovatus (SEQ ID NO:8) and a GH74 xyloglucanase fromTrichoderma reesei.

As any given natural plant biomass is likely to comprise a mixture ofdifferent polysaccharides, sometimes it may be the case that a mixtureof different enzymes is beneficial. Such a mixture may comprise one ormore of any other enzyme. For example, such a mixture might comprise anLPMO with an endo-glucanase, a xylanase with a lichenase, acellobiohydrolase with a mannanase, or an endo-glucanase with acellobiohydrolase in which the enzyme partners are present in molarratios, for example, from 1:100 to 100:1.

In certain cases, the one or more enzymes may be a cocktail of differentenzymes, for example, a crude or semi-crude enzyme preparation. The term“crude enzyme preparation” as used herein generally refers to a solublepreparation extracted from a microbial fermentation that has undergoneminimal processing after the extraction, for example, typically thepreparation may only undergo filtration in order to remove insolublecomponents. The term “semi-crude enzyme preparation” as used hereingenerally refers to a soluble preparation extracted from a microbialfermentation that has undergone some processing after the extraction,for example, the preparation may undergo filtration in order to removeinsoluble components, increasing the enzyme concentration and/ornanofiltration to remove small molecular weight compounds.

In certain cases, the crude or semi-crude enzyme preparation may be froma bacteria or a fungus. In some embodiments, the crude or semi-crudeenzyme preparation may be from a fungus, such as a filamentouscellulolytic fungus, such as from Trichoderma or Aspergillus species. Incertain embodiments, the enzyme may be a crude or semi-crude enzymepreparation from a Trichoderma reesei strain.

In step (e), the one or more oligosaccharides formed in step (d) areremoved, which may be done in a number of ways. They may be isolatedbased on solubility, so that a composition of soluble saccharides onlyis extracted for further processing, and/or isolated chromatographicallyto produce a composition with a narrower band of oligosaccharide chainlengths. Isolation may, for example, be based on precipitation,size-exclusion chromatography, ion-exchange chromatography, filtration,ultrafiltration, microfiltration, or nanofiltration. In the case thatisolation based on solubility is carried out, the profile of saccharidespresent in the isolated composition may depend on the original enzymaticreaction, as different saccharides decrease in solubility with length atdifferent rates.

Also envisaged in the scope of the present disclosure is the furthertreatment of all or part of the removed one or more oligosaccharides toproduce further products before combining them with the one or moredissolved polysaccharides to form the ingredient. This further treatmentmay comprise any chemical, physical, or enzymatic step, such asreduction, for example, reductive amination where suitable; oxidation,caramelization, modification with a Schiff base, or via the Maillardreaction, or by any combination of such steps, and may provide differentproducts having properties which are improved for the desired purpose.For example, the caramelization properties, calorific value, flavor, andcolor may be modified. The oligosaccharides may also be purified, forexample, through precipitation, size-exclusion chromatography,ion-exchange chromatography, filtration, ultrafiltration,microfiltration, or nanofiltration.

Also envisaged in the scope of the disclosure is the further treatmentof all or part of the dissolved one or more soluble polysaccharides toproduce products with improved properties before combining with the oneor more removed oligosaccharides to form the ingredient. This furthertreatment may comprise any chemical, physical, or enzymatic step, suchas alkylation or acid-treatment. The polysaccharides may also bepurified, for example, through precipitation, size-exclusionchromatography, ion-exchange chromatography, filtration,ultrafiltration, microfiltration, or nanofiltration.

In certain cases, following modification and/or purification of theoligosaccharides and polysaccharides, all or part of them are thencombined, as in step (f), which may be at a ratio of from 1:100 to 1:1polysaccharide:oligosaccharide, from 1:10 to 1:1, from 1:90 to 1:2, from1:80 to 1:3, from 1:70 to 1:4, or from 1:60 to 1:5. The specific ratiocan depend on the desired properties of the final ingredient as well asthe modifications and purifications that have been applied to thesaccharides. In certain embodiments, it is not required to recombine allof the removed oligosaccharides and polysaccharides.

In step (f) the combining can be done in a variety of ways, for example,by mixing a solution comprising all or part of the one or more solublepolysaccharides and a solution and/or suspension comprising all or partof the one or more removed oligosaccharides, which may further bespray-dried, lyophilized, or condensed in some other way. The solublepolysaccharides and the removed oligosaccharides may also be combined bymixing a dry form comprising all or part of the one or more removedoligosaccharides produced by spray-drying, lyophilization, orcondensation in some other way after removing them in step (e), with adry form comprising all or part of the one or more solublepolysaccharides, produced by spray-drying, lyophilization, orcondensation in some other way after removing them in step (c).Alternatively, one of (i) the one or more soluble polysaccharides or(ii) the removed oligosaccharides may be in dry form and the other insolution when they are combined.

When the ingredient is in a dry form, the method may further comprise astep (g) of mixing and dissolving the ingredient in a liquid to form aliquid ingredient. In various cases, the liquid may be an aqueoussolution, such as water.

The ingredient formed in step (f), and the liquid ingredient formed instep (g) and of the second aspect of the disclosure, may comprisevarious oligosaccharides and at varying amounts depending on the desiredproperties. In some cases, the ingredient and liquid ingredient maycomprise at least 20% by dry weight or at least 30% by dry weightcello-oligosaccharides having a degree of polymerization of from two tosix, the ingredient may comprise at least 20% by dry weight or at least30% by dry weight xylo-oligosaccharides having a degree ofpolymerization of from two to twelve, the ingredient may comprise atleast 20% by dry weight or at least 30% by dry weight mixed-linkageglucan oligosaccharides having a degree of polymerization of from two tofive, the ingredient may comprise at least 20% by dry weight or at least30% by dry weight manno-oligosaccharides having a degree ofpolymerization of from two to twelve, the ingredient may comprise atleast 20% by dry weight or at least 30% by dry weight xyloglucanoligosaccharides having a degree of polymerization of from four totwelve, and/or the ingredient may comprise at least 20% by dry weight orat least 30% by dry weight chito-oligosaccharides having a degree ofpolymerization of from two to twelve. In some instances, the ingredientcan comprise a maximum of 100% by dry weight of the aboveoligosaccharides and the polysaccharides described herein, therefore theabove embodiments, wherein the oligosaccharides are present in at least20% by dry weight, does not comprise five or six types ofoligosaccharides.

The ingredient and liquid ingredient may comprise at least 50%, 60%,70%, 80%, 90%, 95%, 99%, or 99.5% by dry weight of saccharide present.The ingredient and liquid ingredient may consist essentially ofsaccharides. For example, the ingredient may have less than 0.5% by dryweight or less than 0.3% by dry weight, for instance, 0.1% by dryweight, of other substances.

In certain cases, the liquid ingredient can have a viscosity of from 5to 100,000 cps, from 10 to 80,000 cps, from 20 to 60,000 cps, from 30 to40,000 cps, from 40 to 20,000 cps, or from 50 to 10,000 cps. When theliquid ingredient is to be included into beverages, low syrupviscosities may be desirable, such as about 20 to 300 cps, 50 to 200cps, or 100 to 150 cps. Higher viscosity values may be desired inapplications such as chocolate making, thus where the liquid ingredientis desired to be in a syrup it may have a viscosity in the range ofabout 8,000 to 100,000, about 10,000 to 50,000 cps, or about 15,000 to25,000 cps. The viscosity values are in accordance with testing using aBrookfield HDB VE roto-viscometer using standard testing procedures,wherein a 400 mL sample was taken in a tall-form beaker to ensure thatno container effects occur. The instrument is operated as per themanufacturer's instructions with respect to ranges (rotoviscometry usingspindle code 61, spindle speed 100 rpm, and at 22° C.).

In some instances, the liquid ingredient can have a flow rate of 100 to350 seconds, 150 to 300 seconds, or 200 to 250 seconds. “Flow rate,” asused herein, generally refers to the volume of fluid which passes perunit time. The flow rate values specified herein, unless indicatedotherwise, are determined by measuring by timing the flow rate of 5 mLof the liquid ingredient from a vertically stood syringe (BD Plastipak300613) filled with 20 mL of test liquid under gravity at roomtemperature.

In various cases, the liquid ingredient may have a concentration ofoligo-saccharides of from 1 to 200% w/v, 10 to 150% w/v, 20 to 140% w/v,30 to 130% w/v, 40 to 120% w/v, 50 to 115% w/v, or 60 to 110% w/v.

In some cases, the liquid ingredient may have a concentration ofpolysaccharides of from 0.1 to 50% w/v, 0.2 to 40% w/v, 0.3 to 30% w/v,0.5 to 20% w/v, or 1 to 20% w/v.

In certain cases, the liquid ingredient may have a concentration of thetotal of oligosaccharides and polysaccharides of from 1 to 200% w/v, 10to 160% w/v, 20 to 150% w/v, 30 to 140% w/v, 40 to 130% w/v, 50 to 120%w/v, or 60 to 110% w/v. In various cases, the higher the concentrationof the oligosaccharides and polysaccharides in the liquid, the thickerand more viscous the liquid may become. The liquid ingredient may be ahomogeneous solution.

In another aspect, the ingredient and liquid ingredient may comprise atleast two of the oligosaccharides. The amounts of each of theoligosaccharides may be varied depending on the desired properties ofthe resulting foodstuff, cosmetic, or nutraceutical. The twooligosaccharides may be present in a ratio of 1:9 to 9:1 or 1:2 to 2:1.Further, the ingredient and liquid ingredient may comprise three of theoligosaccharides, they may comprise four oligosaccharides, they maycomprise five oligosaccharides, or they may comprise sixoligosaccharides.

The ingredient and liquid ingredient can comprise thecello-oligosaccharides, for example, cello-oligosaccharides incombination with the xylo-oligosaccharides. Alternatively, theingredient and liquid ingredient can comprise the cello-oligosaccharidesin combination with the manno-oligosaccharides.

The one or more soluble polysaccharides in the ingredient may beparticularly soluble in water or alkali. For example, solublepolysaccharides used in the disclosure can include hemicelluloses suchas xylans, mannans, mixed-linkage glucans, and certain cellulosederivatives such as cellulose acetate, hydroxyethylcellulose, andhydroxymethylcellulose, and chitosan. In some embodiments, the one ormore soluble polysaccharides can comprise hemicellulose. In certainembodiments, the hemicellulose can comprise xylan and/or mannan.

In some instances, the ingredient and liquid ingredient can comprise atleast 2% by dry weight or at least 3% by dry weight of xylan. In variousinstances, the ingredient and liquid ingredient can comprise at least 2%by dry weight or at least 3% by dry weight of mannan. In certaininstances, the ingredient and liquid ingredient can comprise at least 2%by dry weight or at least 3% by dry weight of cellulose derivative. Insome cases, the ingredient and liquid ingredient can comprise at least2% by dry weight or at least 3% by dry weight of mixed-linkage glucan.In various cases, the ingredient and liquid ingredient can comprise atleast 2% by dry weight or at least 3% by dry weight of xyloglucan. Incertain cases, the ingredient and liquid ingredient can comprise atleast 2% by dry weight or at least 3% by dry weight of chitosan.

In certain cases, the ingredient and liquid ingredient can comprise offrom 2 to 40% by dry weight of the one or more soluble polysaccharides,which includes polysaccharide derivatives, from 3 to 30% by dry weightof the one or more soluble polysaccharides, from 5 to 25% by dry weightof the one or more soluble polysaccharides, or from 8 to 20% by dryweight of the one or more soluble polysaccharides.

In some cases, the ingredient and liquid ingredient the ratio ofpolysaccharide:oligosaccharide is from 1:100 to 1:1, from 1:10 to 1:1,from 1:90 to 1:2, from 1:80 to 1:3, from 1:70 to 1:4, or from 1:60 to1:5.

The produced ingredient and liquid ingredient may be useful inapplications in which oligosaccharides, sugar, bulking sweeteners,low-intensity sweeteners, or other related food ingredients areconventionally used. For example, as sweeteners, bulking agents, addeddietary fiber, or humectants. Of particular note is the use of reducingcane sugar in food products. It may be incorporated into cakes, bread,or other baked goods; chocolate or other confectionery such as toffee,fudge, meringue, jam, jelly or caramel; or drinks, for example, toprovide favorable taste or color characteristics or to increase dietaryfiber content. Or the ingredient may be incorporated into animal feed,for example, either as an isolated ingredient or by utilizing theenzymatic reaction mixture directly as feed.

Compositions or ingredients as described herein may be used to alter oneor more properties of a finished product. Such properties include, butare not limited to, sweetness, texture, mouthfeel, binding, glazing,smoothness, moistness, viscosity, color, hygroscopicity, flavor,bulking, water-retention, caramelization, surface texture,crystallization, structural properties, reduced calories, reducedglycemic index, reduced glycemic load, increased fiber, reduced sugar,and dissolution. These may be improvements over what is currentlypossible with saccharides of different types, sugar substitutes, and/orother such compounds.

In the cosmetics industry, the ingredient may improve texture andmoisture retention, act as UV-absorbing molecules, maintain a gel orcream structure, and/or serve as bulking agents. Furthermore, theingredient and liquid ingredient may be useful in nutraceuticalcompositions, as the dietary fiber it provides has been shown toencourage digestive health, well-regulated gut flora, and other benefitsto wellbeing. In this context the ingredients provided herein may alsofunction as an ingredient in a probiotic drink or other prebiotic orprobiotic formulation.

The detailed description is further supplemented with reference to thefollowing numbered embodiments. 1) A method for producing a foodstuff,cosmetic, or a nutraceutical ingredient comprising one or moreoligosaccharides and one or more soluble polysaccharides comprising thesteps of: (a) providing a plant biomass comprising one or more solublepolysaccharides and one or more insoluble polysaccharides; (b) treatingthe plant biomass to dissolve the one or more soluble polysaccharides;(c) removing a portion of the dissolved one or more solublepolysaccharides; (d) reacting the remaining plant biomass with one ormore enzymes to form one or more oligosaccharides; (e) removing the oneor more oligosaccharides; and (f) combining the portion of the dissolvedone or more soluble polysaccharides from step (c) and the one or moreoligosaccharides from step (e) to form the ingredient. 2) The methodaccording to numbered embodiment 1, wherein the treating in step (b) isthermochemical treatment. 3) The method according to numbered embodiment2, wherein the thermochemical treatment is hot water treatment or hotalkali treatment. 4) The method according to numbered embodiment 3,wherein the alkali treatment uses an alkali with a pH of from 10 to 14.5) The method according to either numbered embodiment 3 or 4, whereinthe alkali treatment uses sodium hydroxide, potassium hydroxide, sodiumcarbonate, calcium carbonate, calcium hydroxide, ammonium sulfate,ammonium hydroxide, and aqueous ammonia. 6) The method according to anypreceding numbered embodiment, wherein the treating in step (b) occursat a temperature of from 30 to 180° C. 7) The method according to anypreceding numbered embodiment, wherein the treating in step (b) occursfor 10 minutes to 24 hours. 8) The method of any preceding numberedembodiment, wherein after the removing of the one or moreoligosaccharides, the one or more oligosaccharides and/or dissolved oneor more soluble polysaccharides undergo chemical, physical, or enzymatictreatment, such as reduction, oxidation, caramelization, or Maillardreaction. 9) The method of any preceding numbered embodiment, whereinthe dissolved one or more soluble polysaccharides and/or the one or moreoligosaccharides are dried before being combined together in step (f).10) The method according to numbered embodiment 9, wherein the methodfurther comprises a step: (g) mixing and dissolving the ingredient in aliquid to form a liquid ingredient, wherein the liquid ingredient has aviscosity of from 5 to 100,000 cps. 11) The method according to numberedembodiment 10, wherein the concentration of the oligosaccharides andpolysaccharides in the liquid ingredient is of from 1 to 200% w/v. 12)The method according to any preceding numbered embodiment, wherein theone or more soluble polysaccharides comprise at least one selected fromthe group consisting of: mannans, mixed-linkage glucans, lignocellulose,hemicellulose, certain cellulose derivatives such as cellulose acetate,hydroxyethylcellulose and hydroxymethylcellulose, and chitosan. 13) Themethod according to numbered embodiment 12, wherein the hemicellulosecomprises a xylan and/or a mannan. 14) The method according to anypreceding numbered embodiment, wherein the plant biomass comprises asugar cane biomass, a corn biomass, a wheat biomass, a hardwood, or asoftwood. 15) A foodstuff, cosmetic, or a nutraceutical ingredientobtainable by the method of any preceding numbered embodiment. 16) Afoodstuff, cosmetic, or nutraceutical liquid ingredient comprising atleast one oligosaccharide selected from the list consisting of: i)cello-oligosaccharide having a degree of polymerization of from two tosix; ii) xylo-oligosaccharide having a degree of polymerization of fromtwo to twelve; iii) manno-oligosaccharide having a degree ofpolymerization of from two to twelve; iv) mixed-linkage glucanoligosaccharide having a degree of polymerization of from two to five;v) xyloglucan oligosaccharide having a degree of polymerization of fromfour to twelve; and vi) chito-oligosaccharide having a degree ofpolymerization of from two to twelve; and at least one polysaccharideselected from the list consisting of: i) xylan; ii) mannan; iii)cellulose derivative; iv) mixed-linkage glucan; v) xyloglucan; and vi)chitosan; wherein the liquid ingredient comprises at least 20% by dryweight of the at least one oligosaccharide and at least 2% by dry weightof the at least one polysaccharide, and wherein the liquid ingredienthas a viscosity of from 5 to 100,000 cps, 8,000 to 100,000 cps, 10,000to 50,000 cps, or 15,000 to 25,000 cps. 17) The liquid ingredient ofnumbered embodiment 16, wherein the liquid ingredient comprises at leasttwo of the oligosaccharides listed in (i) to (vi). 18) The liquidingredient of either numbered embodiment 16 or numbered embodiment 17,wherein the liquid ingredient comprises at least 20% by dry weight ofthe cello-oligosaccharides having a degree of polymerization of from twoto six. 19) The liquid ingredient of any one of numbered embodiments 16to 18, wherein the liquid ingredient comprises at least 20% by dryweight of the xylo-oligosaccharide having a degree of polymerization offrom two to twelve. 20) The liquid ingredient of any one of numberedembodiments 16 to 19, wherein the liquid ingredient comprises at least20% by dry weight of the manno-oligosaccharide having a degree ofpolymerization of from two to twelve. 21) The liquid ingredient of anyone of numbered embodiments 16 to 20, wherein the liquid ingredientcomprises at least 2% by dry weight of the xylan. 22) The liquidingredient of any one of numbered embodiments 16 to 21, wherein theliquid ingredient comprises at least 2% by dry weight of the mannan. 23)The liquid ingredient of any one of numbered embodiments 16 to 22,wherein the liquid ingredient comprises at least 2% by dry weight of thecellulose derivative. 24) The liquid ingredient of any one of numberedembodiments 16 to 23, wherein the liquid ingredient has a concentrationof polysaccharides of from 0.1 to 50% (w/v). 25) The liquid ingredientof any one of numbered embodiments 16 to 24, wherein the liquidingredient has a concentration of oligosaccharides of from 1 to 200%(w/v). 26) The liquid ingredient of any one of numbered embodiments 16to 25, wherein the liquid ingredient has a concentration ofpolysaccharides and oligosaccharides of from 1 to 200% (w/v). 27) Theliquid ingredient of any one of numbered embodiments 16 to 26, whereinthe liquid ingredient comprises an amount of polysaccharide andoligosaccharide in a ratio from 1:100 to 1:1. 28) The liquid ingredientof any one of numbered embodiments 16 to 27, wherein the liquidingredient comprises two oligosaccharides in a ratio from 1:9 to 9:1 inrelation to each other. 29) Use of the liquid ingredient of any ofnumbered embodiments 16 to 28 in a foodstuff, cosmetic, or nutraceuticalproduct.

Additional Exemplary Embodiments of Pretreating Biomass to RemoveMonosaccharides and/or Disaccharides

In some cases, the present disclosure relates to novel methods ofphysically and thermochemically treating plant biomass materials for theproduction of foodstuff, cosmetic, or nutraceutical ingredients.

Sugary foods and drinks are an important part of culture and lifestylehabits across the world, but the sugar they contain has been linked toobesity, diabetes, poor dental health, and disruptive behavior inpeople. Because of this, consumer preferences have been shifting awayfrom sugar-containing foods, and governments are increasinglyimplementing regulation to encourage the consumption of less sugar.

As such, industry has been searching for suitable low-calorie sweetenersfor many decades to substitute for sugar in food and beverages.Unfortunately, many sugar substitutes are produced from non-naturalresources, and often offer bitter undertones or other unpleasant tastesalong with their sweetness, both of which consumers find unappealing.Moreover, while many sweeteners are able to mimic the sweetness of sugarin food and drinks, few are able to mimic the broad range of roles thatsugar plays in food, such as adding bulk, modulating texture, providingstructure, acting as a preservative, and modulating color and flavorthrough caramelization and Maillard reactions.

Dietary fiber is an important part of a positive diet and helps maintaindigestive health and a well-regulated gut flora. Such fiber comprisessaccharides of varying chain lengths and types. In addition to beingfound naturally in a wide spectrum of foods, fiber can also be producedseparately and added to other foods during their manufacture.

Biomass is a good source of saccharides that can be used to replacesugar and add fiber to food products. Compositions made from feedstockshave been provided. However, there remains a need to optimize theprocess by which these saccharides are obtained from the biomass andprocessed into the compositions useful as a foodstuff, cosmetic, ornutraceutical ingredient on a large and commercial scale. Enzymebreakdown of a large amount of plant biomass can take a considerableamount of time. Furthermore, the saccharides generally need to beproduced by controlled breakdown. Enzyme breakdown can be desirable forthis as product sizes can be exquisitely controlled, ensuring no and/orlittle monosaccharides are yielded.

Methods, as provided herein, can economically and efficiently enable theproduction of a foodstuff, cosmetic, or nutraceutical ingredient from aplant biomass starting material which is quicker than previously usedmethods, yields a purer final product, and can be used on a large andcommercial scale. The method can do so by performing a prewashing stepto remove from biomass endogenous monosaccharides and/or disaccharides,and employing thermochemical pretreatment steps that may ensurecontrolled breakdown and release of the saccharides that can be neededto manufacture the ingredient. Together, these steps may ensure that noand/or little monosaccharides are yielded during pre-enzyme processing.This can maximize efficiency and limit the amount of post-reactionpurification needed.

Accordingly, in another aspect of the disclosure there is provided amethod for producing a foodstuff, cosmetic, or nutraceutical ingredient,the ingredient comprising one or more oligosaccharides, wherein themethod comprises the steps of:

-   -   a) a physical pretreatment of a plant biomass comprising        monosaccharides and/or disaccharides;    -   b) a washing cycle (also referred to herein as an incubation        cycle) comprising the steps of (i) washing (e.g., incubating)        the plant biomass to solubilize at least a portion of the        monosaccharides and/or disaccharides and (ii) removing the at        least a portion of monosaccharides and/or disaccharides;    -   c) a thermochemical pretreatment of the plant biomass;    -   d) forming the one or more oligosaccharides by an enzymatic        reaction, the enzymatic reaction comprising the step of        contacting, in a solution or suspension, one or more        polysaccharide-cleaving enzymes and the plant biomass;    -   e) separating (also referred to herein as enriching for or        isolating) the one or more oligosaccharides from the enzymatic        reaction mixture and using the one or more oligosaccharides to        form the ingredient.

Preparing the foodstuff, cosmetic, or nutraceutical ingredient in themanner provided herein can allow for: efficient use of biomass byincorporating oligomeric and polymeric material from the same biomasssource, purification, derivatization or other modification, as well ascontrol of oligomeric and polymeric proportions, which can improve thefunctional properties, nutritional properties, and tolerance of theingredient.

Steps (a), (b), and (c) of the method of the disclosure are all“pretreatment” steps which are performed on the plant biomass startingmaterial. “Pretreatment,” as used herein, generally refers to steps thatare performed on the plant biomass before a polysaccharide-cleavingenzyme is put into contact with the plant biomass.

Step (a) is a physical pretreatment of the plant biomass, which can havethe purpose of physically breaking down the plant biomass in preparationfor the subsequent steps. The physical step may help speed up theoverall method because it may increase the available surface area of theplant biomass enabling the chemicals used in subsequent steps to beactive on more of the plant biomass, for example, at one time. Thephysical pretreatment step may comprise chipping, chopping, milling,ball-milling, grinding, sprucing, blending, or a combination thereof, ofthe plant biomass.

Any substance which comprises suitable polysaccharides may be the plantbiomass. As the foodstuff, cosmetic, and nutraceutical industries use abroad variety of oligosaccharides, the polysaccharides suitable in themethod are not particularly limited. Plant biomass suitable forproducing the oligosaccharide profile of the current disclosure maycomprise, for example, cellulose, lignocellulose, chitin, chitosan,xylan (such as glucuronoxylan, arabinoxylan, and glucuronoarabinoxylan)xyloglucan, and mixed-linkage glucan, and/or mannan (such asglucomannan, galactomannan, or galactoglucomannan). However, any plantbiomass which can be suitably acted upon is envisaged.

As such, the plant biomass may be grain, grain chaff, bean pods, seedcoats, and/or other seed materials; seaweeds; corn stover, straw,bagasse, miscanthus, sorghum residue, switch grass, bamboo, and/or othermonocotyledonous tissue; water hyacinth, leaf tissue, roots, and/orother vegetative matter; and/or any combination of suitable plantbiomasses. In some embodiments, the plant biomass can comprise sugarcane, corn stover, corncob, wheat bran, wheat straw, hardwood orsoftwood. In certain embodiments, the plant biomass can comprisecorncob.

Step (b) is a washing cycle (or incubation cycle) pretreatment of theplant biomass that can occur after the physical pretreatment step. Theaim of step (b) may be to solubilize and remove monosaccharides and/ordisaccharides from the biomass. For example, the monosaccharides and/ordisaccharides may include, but are not limited to, free sucrose,maltose, lactose, glucose, fructose, or galactose. Removal of freedisaccharides such as sucrose can be of interest, as disaccharidescannot subsequently be easily removed from the oligosaccharide fraction,for example, filtration methods can be used but would generally causeequal loss of other disaccharides.

Step (i) of the washing cycle may occur at a range of temperatures, forexample, of from 5 to 150° C., from 10 to 100° C., or from 15 to 50° C.In some cases, the washing cycle can occur at room temperature, forinstance, at about 15 to 25° C. or about 20 to 22° C. Highertemperatures may allow for quicker solubilization of the monosaccharidesand/or disaccharides, however, too high temperatures can be moredifficult to achieve in an efficient and cost-effective manner and maydamage the biomass compounds or solubilize compounds that are notdesirable to be solubilized during this step.

Step (i) of the washing cycle may occur for a range of time scales, forexample, large amounts of biomass may be exposed to this step for alonger period of time, which can be adjusted accordingly. For example,the time scale may be of from 0.5 minutes to 72 hours, from 1 minute to12 hours, from 5 minutes to 24 hours, or from 10 minutes to 3 hours. Incertain embodiments, this step may occur as batch or continuous.

In some embodiments, step (i) of the washing cycle may comprise washingthe plant biomass at room temperature in water, i.e., water supplied ata neutral pH of about pH 7. In another aspect, the step (i) of thewashing cycle may comprise heating the plant biomass in water, i.e.,water supplied at a neutral pH of about pH 7. During the washing cycle,the neutral water supplied may become slightly acidic as monosaccharidesand/or disaccharides are solubilized.

In certain embodiments, step (i) of the washing cycle may compriseheating the plant biomass in an alkali solution having a pH of from 7.1to 14, from 7.5 to 12, or from 8 to 11. The solution may comprise, orsuitably consist of, any one of the alkalis selected from: sodiumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,calcium hydroxide, ammonium sulfate, ammonium hydroxide, and aqueousammonia. In various embodiments, the alkali may be sodium hydroxide. Acombination of the listed alkalis is also envisaged.

In various cases, step (i) of the washing cycle may comprise heating theplant biomass in an acidic solution having a pH of from 1 to 6.9, from 2to 6.5, or from 4 to 6. The solution may comprise, or suitably consistof, any organic or mineral acids, such as one of the acids selectedfrom: sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid,acetic acid, maleic acid, fumaric acid, and oxalic acid. In certaincases, the acid may be sulfuric acid. A combination of the listed acidsis also envisaged.

In certain instances, a portion or all of the monosaccharides and/ordisaccharides in the plant biomass are solubilized and removed duringstep (b). Other contaminants may also be removed during this step, suchas other soluble sugars and minerals.

Step (b) may be repeated to remove monosaccharides and/or disaccharidesthat were not removed from the plant biomass during the washing cycle.In various instances, step (b) may be performed at least two times, atleast three times, at least four times, or at least five times. Step (c)is a thermochemical pretreatment of the plant biomass that can occurafter the physical and washing pretreatment steps. “Thermochemical,” asused herein, generally refers to heating above room temperature (roomtemperature is, for instance, about 15 to 25° C. or about 20 to 22° C.)the plant biomass in a chemical, such as heating in a solution of water,acid, or alkali. The purpose of the thermochemical step can be to helpspeed up the overall method because it may chemically modify chemicalcomponents of the plant biomass, for example, it can disrupt hydrogenbonds between saccharides enabling the enzyme in the subsequent step tomore easily break up the saccharides.

The heating may be at a range of temperatures, for example, from 50 to150° C., from 60 to 130° C., from 65 to 120° C., or from 70 to 110° C.Higher temperatures can allow for quicker chemical and/or physicalmodification, however, too high temperatures can be more difficult toachieve in an efficient and cost-effective manner.

The heating may occur for a range of time scales, particularly largeamounts of biomass may be exposed to heating for a longer period oftime, which can be adjusted accordingly. For example, the heating of theplant biomass can be of from 5 minutes to 72 hours, from 15 minutes to24 hours, from 30 minutes to 12 hours, or from 1 hour to 4 hours.

In some embodiments, the thermochemical pretreatment may compriseheating the plant biomass in water, i.e., at a neutral pH of about pH 7.

In certain embodiments, the thermochemical treatment may compriseheating the plant biomass in an alkali solution having a pH of from 7.1to 14, from 9 to 13, or from 10 to 13. The solution may comprise, orsuitably consist of, any one of the alkalis selected from: sodiumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,calcium hydroxide, ammonium sulfate, ammonium hydroxide and aqueousammonia. In various embodiments, the alkali may be sodium hydroxide. Acombination of the listed alkalis is also envisaged.

In some cases, the thermochemical treatment may comprise heating theplant biomass in an acidic solution having a pH of from 1 to 6.9, from 2to 6.5, or from 4 to 6. The solution may comprise, or suitably consistof, any organic or mineral acids, such as one of the acids selectedfrom: sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid,acetic acid, maleic acid, fumaric acid, and oxalic acid. In certaincases, the acid may be sulfuric acid. A combination of the listed acidsis also envisaged.

Multiple different sequential thermochemical treatment steps are alsoenvisaged for step (c). For example, step (c) may be performed at leasttwo times, at least three times, at least four times, or at least fivetimes.

In certain cases, the washing of step (b) may occur in water and thepretreatment of step (c) may occur in alkali.

After the pretreatment steps, step (d) may comprise the enzymaticreaction forming the one or more oligosaccharides from the plantbiomass. The polysaccharides present in the plant biomass may bepartially cleaved by enzymes into oligosaccharides (e.g., usefuloligosaccharides), leaving partially cleaved, or uncleaved,polysaccharides, which may include cellulose, xylan (such asglucuronoxylan, arabinoxylan, or glucuronoarabinoxylan), mannan (such asglucomannan, galactomannan, or galactoglucomannan), mixed-linkageglucan, xyloglucan chitin, chitosan, or lignocellulose.

The reaction may take place in solution and/or suspension. The reactionmay take place in a suitable reaction vessel. In some cases, thereaction may take place at a temperature or temperature protocolsuitable for the particular combination of enzyme and plant biomass, thereaction may be allowed to progress for a certain amount of time, untilthe products have reached a desired concentration, or until some otherrequirement has been met and the products are isolated or otherwisecollected. This time period may be from about 1 minute to about 6 days,from about 0.5 days to about 5 days, or from about 16 hours to about 96hours. The reaction may alternatively be allowed to proceed until nofurther catalysis occurs.

In order to ensure optimal contact between the enzymes and the plantbiomass, the reaction mixture may be agitated, either constantly or atintervals. The agitation may take the form of rhythmically moving theentire reaction vessel, of a fan or other stirring device, of a bubblesparging, or any other method of agitation.

The enzymatic reaction may be a microbial fermentation. The temperatureand reaction time may be suitable for the growth of the microbialorganism used. The microbial organism may be genetically altered toproduce an enzyme suitable for the production of an oligosaccharide ofthe present disclosure. The microbe may be, for example, a bacterium,for example, Escherichia coli, or a fungus, such as Saccharomycescerevisiae or Trichoderma reesei.

Further embodied in the present disclosure is an expression vectorsuitable for modifying the subject microorganism such that it producesan enzyme or mixture of enzymes of the current disclosure. Wheredesired, the expression vector, which may be a plasmid or any othernucleic acid able to induce production of the enzyme, may comprise oneor more of the following regulatory sequences so as to control theexpression of the exogenous enzyme: regulatory sequences of a heat shockgene, regulatory sequences of a toxicity gene, and regulatory sequencesof a spore formation gene.

The enzymatic reaction can be carried out at a temperature ortemperature protocol suitable to the enzymes and substrates used. Forexample, it may be carried out at a constant temperature in the range offrom about 10° C. to about 100° C., about 20° C. to about 70° C., orabout 30° C. to about 40° C. If the enzymatic reaction takes the form ofa microbial fermentation, the temperature may be suitable for such, forexample, the enzymatic reaction may comprise the growth of E. coliand/or the temperature may be constant and about 37° C.

The pH of the solution or suspension may affect the activity of theenzymes. Control of pH may assure that an enzymatic reaction proceeds ata suitable rate. The enzymatic reaction of the present disclosure maytake place at a pH in the range of from about 2 to about 10, about 3 toabout 8, or about 4 to about 6.

The enzymatic reaction may be allowed to continue to run until there is5-75% undigested polysaccharide-containing plant biomasses remaining,5-70%, 5-65%, 5-55%, more or 10-50%. This can be monitored or checked byreducing end assays, such as the anthrone assay and/or bychromatographic methods such as thin-layer chromatography andhigh-performance anion exchange chromatography.

There are many enzymes that may be suitable for use in the enzymaticreaction of the present method. For example, “lytic polysaccharidemonooxygenase” and “LPMO” which are a class of enzymes able tooxidatively cleave polysaccharides using a copper comprising moiety andusing an oxygen source, such as a molecule of dioxygen, peroxide, or anyother oxygen source; and a suitable reducing agent. As such, when anLPMO is used, the enzymatic reaction may be carried out under aerobicconditions. Suitable reducing agents are not particularly limited, butexamples include ascorbic acid, gallic acid, cysteine, NADH, NADPH,pyrogallol, dithiothreitol, cyanoborohydrides, borohydrides,photosynthetic pigments, lignin, lignols, and a combination ofcellobiose and cellobiose dehydrogenase. A wide variety ofphotosynthetic pigments may be used, for example, thylakoids andpurified fractions or chlorophyllin and light may be supplied. LPMOs canbe selected from the following families: AA9, AA10, AA11, AA13, AA14 andAA15. The LPMO may be PaLPMO9E (SEQ ID NO:1), an AA9 LPMO originallyisolated from the ascomycete fungus Podospora anserina. The LPMO may bean AA9 LPMO from Trichoderma reesei (SEQ ID NO:23).

Aerobic conditions may comprise the addition of oxygen, which may beprovided by aeration of the substrate mixture with an oxygen-comprisinggas, such as air. Aeration may be conducted by the introduction ofoxygen-comprising air bubbles into the aqueous substrate mixtures byvarious systems, such as an air-injector, an aeration frit, a membranesystem, or an internal-loop airlift reactor. The concentration ofmolecular oxygen in the enzymatic reaction may be from about 4 mg/L toabout 14 mg/L.

Another type of enzyme that can be used in the method is a “cellulase”which has hydrolytic activity against cellulose, for example,endo-1,4-beta-glucanase, cellobiohydrolase, and/or beta-glucosidaseactivities. Such enzymes are able to cleave glycosidic bonds in one ormore forms of cellulose, including cellulose found in plant biomass. Indoing so they produce products including glucose andcello-oligosaccharides. Beta-glucanases include enzymes from GH5, GH7,and GH12 enzyme, such as those derived from Aspergillus niger (SEQ IDNOs:12, 13, and 14) and Trichoderma reesei (SEQ ID NOs:24 and 25).

Another type of enzyme is “cellobiohydrolase” that has hydrolyticactivity against cellulose, and produces mainly cellobiose as a product.Cellobiose is a disaccharide, and is a cello-oligosaccharide. Suchenzymes are able to cleave glycosidic bonds in one or more forms ofcellulose, including cellulose found in plant biomass.Cellobiohydrolases may be from GH6 and GH7 enzyme families or Cel6A orCel7A enzymes derived from Trichoderma reesei (SEQ ID NOs:10 and 11).

Another type of enzyme is “beta-glucosidase” that has hydrolyticactivity against cellulose, and produces mainly glucose as a product.Such enzymes are able to cleave glycosidic bonds in one or more forms ofcellulose, including cellulose found in plant biomass. Beta-glucosidasesmay include GH3 beta-glucosidases, such as from Trichoderma reesei (SEQID NO: 22).

Another type of enzyme is a lichenase, which may be selected from: theGH5, GH7, GH8, GH9, GH12, GH16, GH17, or GH26 families. In some cases,the lichenase may be a GH16 enzyme. The GH16 enzyme may be derived fromBacillus subtilis (SEQ ID NO:2). The enzyme is able to act on, forexample, mixed-linkage glucans, which are glucans comprising a mixtureof β-1,3 and β-1,4 linkages, and may cleave them at β-1,4 glycosidicbonds. In the case in which the lichenase acts on a mixed-linkageglucan, the β-glucans produced may fall largely within the size range offrom about 3 to about 7 residues, so they may be useful in the food,cosmetics, and nutraceutical industries. Mixed-linkage glucans areabundant in members of the grass and horsetail families, and as such,grass-based biomasses such as straw have high levels of it, and may beacted upon usefully with lichenases.

Another type of enzyme is a xylanase, which may act on, for example,plant biomass comprising a xylan backbone. The xylanase may be, forexample, a glucuronoxylanase, an arabinoxylanase, or aglucuronoarabinoxylanase. The enzyme may be active on a variety ofpolymers having a xylan backbone, such as glucuronoxylan, arabinoxylan,and glucuronoarabinoxylan. These polymers are abundant in various plantbiomass, for example, both hardwood and softwood may comprise suitablepolysaccharides, with hardwood often comprising glucuronoxylan andsoftwood often comprising arabinoglucuronoxylan. In some embodiments,xylanases may include GH5 xylanases from Ruminiclostridium thermocellum(SEQ ID NO:3) and Gonapodya prolifera (SEQ ID NO:4), and GH30 xylanasesfrom Dickeya chrysanthemi (SEQ ID NO:5), Bacillus subtilis (SEQ IDNO:6), Bacteroides ovatus (SEQ ID NO:7), and Trichoderma reesei (SEQ IDNO:15).

Other enzymes useful in the disclosure may include xyloglucanases andxyloglucan endoglucanases (XEGs), which are produced by numerousorganisms, including plant-pathogenic microbes. They are able to act onxyloglucan, a hemicellulosic β-1,4 glucan chain abundant in the primarycell wall of higher plants, which is decorated with xylose, some of thexylose residues being further decorated with other residues, such asgalactose. When suitable xyloglucanases or XEGs act on xyloglucan, theproducts comprise xyloglucan oligosaccharides having a main chain of alength that can be useful in the foodstuff, cosmetics, and nutraceuticalindustries. Xyloglucanases can include a GH5 xyloglucanase fromBacteroides ovatus (SEQ ID NO:8) and a GH74 xyloglucanase fromTrichoderma reesei.

As any given natural plant biomass is likely to comprise a mixture ofdifferent polysaccharides, it can sometimes be the case that a mixtureof different enzymes is beneficial. Such a mixture may comprise one ormore of any other enzyme. For example, such a mixture might comprise anLPMO with an endo-glucanase, a xylanase with a lichenase, acellobiohydrolase with a mannanase, or an endo-glucanase with acellobiohydrolase in which the enzyme partners are present in molarratios from 1:100 and 100:1.

In some instances, the one or more enzymes may be a cocktail ofdifferent enzymes, for example, a crude or semi-crude enzymepreparation. The term “crude enzyme preparation,” as used herein,generally refers to a soluble preparation extracted from a microbialfermentation that has undergone minimal processing after the extraction.For example, typically the preparation may only undergo filtration inorder to remove insoluble components. The term “semi-crude enzymepreparation,” as used herein, generally refers to a soluble preparationextracted from a microbial fermentation that has undergone someprocessing after the extraction, for example, the preparation mayundergo filtration in order to remove insoluble components, increasingthe enzyme concentration and/or nanofiltration to remove small molecularweight compounds.

In some cases, the crude or semi-crude enzyme preparation may be from abacteria or a fungus. For example, the preparation may be from a fungus,such as a filamentous cellulolytic fungus, such as from Trichoderma orAspergillus species. The enzyme may be a crude or semi-crude enzymepreparation from a Trichoderma reesei strain.

In step (e), the oligosaccharides may be separated from the enzymaticreaction mixture in a number of ways. They may be isolated based onsolubility, so that a composition of soluble saccharides only isextracted for further processing, and/or isolated chromatographically toproduce a composition with a narrower band of oligosaccharide chainlengths. Isolation may, for example, be based on precipitation,size-exclusion chromatography, ion-exchange chromatography, filtration,ultrafiltration, microfiltration, or nanofiltration. In the case thatisolation based on solubility is carried out, the profile of saccharidespresent in the isolated composition may depend on the original enzymaticreaction, as different saccharides decrease in solubility with length atdifferent rates.

Also envisaged in the scope of the present disclosure is the furthertreatment of all or part of the produced oligosaccharides to producefurther products before incorporation into a foodstuff, cosmetic, ornutraceutical. This further treatment may comprise any chemical,physical, or enzymatic step, such as reduction, for example, reductiveamination where suitable; oxidation, caramelization, modification with aSchiff base, or via the Maillard reaction, or by any combination of suchsteps, and may provide different products having properties which areimproved for the desired purpose. For example, the caramelizationproperties, calorific value, flavor, and color may be modified. Theoligosaccharides may also be purified, for example, throughprecipitation, size-exclusion chromatography, ion-exchangechromatography, filtration, ultrafiltration, microfiltration, ornanofiltration.

The ingredient formed in step (e) may comprise various oligosaccharidesand at varying amounts depending on the desired properties. In variouscases, the ingredient may comprise at least 20% by dry weight or atleast 30% by dry weight cello-oligosaccharides having a degree ofpolymerization of from two to six, the ingredient may comprise at least20% by dry weight or at least 30% by dry weight xylo-oligosaccharideshaving a degree of polymerization of from two to twelve, the ingredientmay comprise at least 20% by dry weight or at least 30% by dry weightmixed-linkage glucan oligosaccharides having a degree of polymerizationof from two to five, the ingredient may comprise at least 20% by dryweight or at least 30% by dry weight manno-oligosaccharides having adegree of polymerization of from two to twelve, the ingredient maycomprise at least 20% by dry weight or at least 30% by dry weightxyloglucan oligosaccharides having a degree of polymerization of fromfour to twelve, and/or the ingredient may comprise at least 20% by dryweight or at least 30% by dry weight chito-oligosaccharides having adegree of polymerization of from two to twelve. In some embodiments, theingredient can comprise a maximum of 100% by dry weight of the aboveoligosaccharides and the polysaccharides described herein, therefore theabove embodiment, wherein the oligosaccharides are present in at least20% by dry weight, does not comprise all six types of oligosaccharides.

In some instances, the ingredient may comprise at least 50%, 60%, 70%,80%, 90%, 95%, 99%, or 99.5% by dry weight of saccharide present. Theingredient may consist essentially of saccharides. For example, theingredient may have less than 0.5% by dry weight or less than 0.3% bydry weight, for instance, 0.1% by dry weight, of other substances.

In various instances, the ingredient may comprise at least two of theoligosaccharides. The amounts of each of the oligosaccharides may bevaried depending on the desired properties of the resulting foodstuff,cosmetic, or nutraceutical. The two oligosaccharides may be present in aratio of 1:9 to 9:1 or 1:2 to 2:1. Further, the ingredient may comprisethree of the oligosaccharides, four of oligosaccharides, five of theoligosaccharides, or six of the oligosaccharides.

In some embodiments, the ingredient may comprise thecello-oligosaccharides, for instance, cello-oligosaccharides incombination with the xylo-oligosaccharides. In certain embodiments, theingredient may comprise the cello-oligosaccharides in combination withthe manno-oligosaccharides.

The produced ingredient can be useful in applications in whicholigosaccharides, sugar, bulking sweeteners, low-intensity sweeteners,or other related food ingredients are conventionally used. For example,as sweeteners, bulking agents, added dietary fiber, or humectants. Ofparticular note may be in the use of reducing cane sugar in foodproducts. The ingredient may be incorporated into cakes, bread, or otherbaked goods; into chocolate or other confectionery such as toffee,fudge, meringue, jam, jelly or caramel; or drinks, for example, toprovide favorable taste or color characteristics or to increase dietaryfiber content. In some instances, the ingredient may be incorporatedinto animal feed, for example, either as an isolated ingredient or byutilizing the enzymatic reaction mixture directly as feed.

Compositions or ingredients as described herein may be used to alter oneor more properties of a finished product. Such properties include, butare not limited to, sweetness, texture, mouthfeel, binding, glazing,smoothness, moistness, viscosity, color, hygroscopicity, flavor,bulking, water-retention, caramelization, surface texture,crystallization, structural properties, reduced calories, reducedglycemic index, reduced glycemic load, increased fiber, reduced sugar,and dissolution. These may be improvements over what is currentlypossible with saccharides of different types, sugar substitutes, and/orother such compounds.

In the cosmetics industry, the ingredient may improve texture andmoisture retention, act as UV-absorbing molecules, maintain a gel orcream structure, and/or serve as bulking agents. Furthermore, theingredient can be useful in nutraceutical compositions, as the dietaryfiber it provides has been shown to encourage digestive health,well-regulated gut flora, and other benefits to wellbeing. In thiscontext the ingredients herein may also function as an ingredient in aprobiotic drink or other prebiotic or probiotic formulation.

The detailed description is further supplemented with reference to thefollowing numbered embodiments. 1) A method for producing a foodstuff,cosmetic, or nutraceutical ingredient, the ingredient comprising one ormore oligosaccharides, wherein the method comprises the steps of: a) aphysical pretreatment of a plant biomass comprising monosaccharidesand/or disaccharides; b) a washing cycle comprising the steps of (i)washing the plant biomass to solubilize at least a portion of themonosaccharides and/or disaccharides and (ii) removing the at least aportion of monosaccharides and/or disaccharides; c) a thermochemicalpretreatment of the plant biomass; d) forming the one or moreoligosaccharides by an enzymatic reaction, the enzymatic reactioncomprising the step of contacting, in a solution or suspension, one ormore polysaccharide-cleaving enzymes and the plant biomass; e)separating the one or more oligosaccharides from the enzymatic reactionmixture and using the one or more oligosaccharides to form theingredient. 2) The method of numbered embodiment 1, wherein the physicalpretreatment step comprises chipping, chopping, milling, ball-milling,grinding, sprucing or blending of the plant biomass. 3) The method ofeither numbered embodiment 1 or numbered embodiment 2, wherein step (i)of the washing cycle occurs in water, acid, or alkali. 4) The method ofany preceding numbered embodiment, wherein step (i) of the washing cycleoccurs at a temperature of from 5 to 150° C., from 10 to 100° C., orfrom 15 to 50° C. 5) The method of any preceding numbered embodiment,wherein step (i) of the washing cycle occurs for a time scale of from0.5 minutes to 72 hours, of from 1 minute to 12 hours, of from 5 minutesto 24 hours, or from 10 minutes to 3 hours. 6) The method of anypreceding numbered embodiment, wherein the thermochemical pretreatmentcomprises heating the plant biomass in a solution of water, acid, oralkali. 7) The method of numbered embodiment 6, wherein the heating ofthe plant biomass is at a temperature of from 50 to 150° C., of from 60to 130° C., of from 65 to 120° C., or of from 70 to 110° C. 8) Themethod of either numbered embodiment 6 or 7, wherein the heating of theplant biomass is of from 5 minutes to 72 hours, from 15 minutes to 24hours, from 30 minutes to 12 hours, or from 1 hour to 4 hours. 9) Themethod of any one of numbered embodiments 6 to 8, wherein the solutionhas a pH of from 7.1 to 14, 7.5 to 12, or of from 8 to 11. 10) Themethod of numbered embodiment 9, wherein the solution comprises sodiumhydroxide potassium hydroxide, sodium carbonate, calcium carbonate,aqueous ammonia, ammonium sulfate, or ammonium hydroxide. 11) The methodof any one of numbered embodiments 6 to 8, wherein the solution has a pHof from 1 to 6.9, of from 2 to 6.5, or of from 4 to 6. 12) The method ofnumbered embodiment 11, wherein the solution comprises sulfuric acid,hydrochloric acid, nitric acid, phosphoric acid, acetic acid, maleicacid, fumaric acid, or oxalic acid. 13) The method of any one ofnumbered embodiments 1 to 12, wherein the plant biomass is sugar cane,corn stover, corncob, wheat bran, wheat straw, hardwood, or softwood.14) The method of any one of numbered embodiments 1 to 13, wherein theplant biomass comprises cellulose, chitin, chitosan, xylan, xyloglucan,mixed-linkage glucan, mannan, or lignocellulose. 15) The method of anyone of numbered embodiments 1 to 14, wherein the one or more of thepolysaccharide-cleaving enzymes is one of cellulase, xylanase,xyloglucanase, endo-glucanase, cellobiohydrolase, mannanase, lichenaseor a lytic polysaccharide monooxygenase (LPMO), for example, selectedfrom the group consisting of: AA9, AA10, AA11, AA13, AA14, and AA15. 16)The method of any one of numbered embodiments 1 to 15, wherein the oneor more of the polysaccharide-cleaving enzymes is prepared from T.reesei fungi. 17) The method of any one of numbered embodiments 1 to 16,wherein the one or more oligosaccharides comprise one of β-glucans,cello-, MLG-, mannan-, or xylo-oligosaccharide. 18) The method of anyone of numbered embodiments 1 to 17, wherein the polysaccharide-cleavingenzyme(s) is operably linked to a catalytic or non-catalytic module, forexample, wherein the polysaccharide-cleaving enzyme is operably linkedto a non-catalytic module and the non-catalytic module is acarbohydrate-binding module. 19) The method of any one of numberedembodiments 1 to 18, wherein after the separating of the one or moreoligosaccharides, the one or more oligosaccharides undergo chemical,physical, or enzymatic treatment, such as reduction, oxidation,caramelization, or Maillard reaction.

The detailed description is further supplemented with reference to thefollowing numbered embodiments. 1) A method for producing an ingredientfor human consumption, the method comprising: (a) physically treating aplant biomass; (b) subjecting the physically treated plant biomass to anincubation cycle comprising: (i) incubating the physically treated plantbiomass in an incubation solution having a pH from 6.6 to 7.4 tosolubilize monosaccharides and/or disaccharides from the physicallytreated plant biomass; and (ii) removing a portion of the solubilizedmonosaccharides and/or disaccharides from the incubation solution; (c)thermochemically treating the incubated plant biomass in one of (i) anacidic solution having a pH from 2 to 6.5 or (ii) an alkali solutionhaving a pH from 7.5 to 12; (d) contacting, in a solution or suspension,one or more polysaccharide-cleaving enzymes and the thermochemicallytreated plant biomass to form one or more oligosaccharides; and (e)enriching the solution or suspension to increase the concentration ofthe one or more oligosaccharides to form the ingredient. 2) The methodof numbered embodiment 1, further comprising removing at least a portionof the monosaccharides and/or disaccharides from the incubation solutionat step (b)(ii). 3) The method any preceding numbered embodiment,wherein the thermochemically treated plant biomass comprises no orsubstantially no monosaccharides. 4) The method of any precedingnumbered embodiment, further comprising purifying the one or moreoligosaccharides from the solution or suspension. 5) The method of anypreceding numbered embodiment, further comprising repeating step (b). 6)The method of numbered embodiment 5, wherein step (b) is conducted two,three, four, or five times. 7) The method of any preceding numberedembodiment, further comprising repeating step (c). 8) The method ofnumbered embodiment 7, wherein step (c) is conducted two, three, four,or five times. 9) The method of any preceding numbered embodiment,further comprising discarding the portion of the solubilizedmonosaccharides and/or disaccharides removed in step (b). 10) The methodof any preceding numbered embodiment, wherein the portion of thesolubilized monosaccharides and/or disaccharides removed in step (b) isnot combined with the portion of the one or more oligosaccharides ofstep (e) to form the ingredient. 11) The method of any precedingnumbered embodiment, wherein the ingredient is substantially free ofmonosaccharides. 12) The method of any preceding numbered embodiment,wherein the ingredient is substantially free of disaccharides. 13) Themethod of any preceding numbered embodiment, wherein the one or moreoligosaccharides comprise at least one of: i) a cello-oligosaccharidehaving a degree of polymerization (DP) of from two to six; ii) axylo-oligosaccharide having a DP of from two to twelve; iii) anarabinoxylo-oligosaccharide having a DP of from three to fifteen; iv) amanno-oligosaccharide having a DP of from two to twelve; v) amixed-linkage glucan oligosaccharide having a DP of from two to five;vi) a xyloglucan oligosaccharide having a DP of from four to twelve; orvii) a chito-oligosaccharide having a DP of from two to twelve. 14) Themethod of numbered embodiment 13, wherein the ingredient comprises atleast two of the oligosaccharides listed in (i) to (vii). 15) The methodof numbered embodiment 14, wherein the ingredient comprises the at leasttwo oligosaccharides in a ratio from 1:9 to 1:1 in relation to eachother. 16) The method of any preceding numbered embodiment, wherein themonosaccharides and/or disaccharides comprise at least one of sucrose,glucose, maltose, lactose, glucose, fructose, or galactose. 17) Themethod of any preceding numbered embodiment, wherein the physicallytreating of step (a) comprises at least one of chipping, chopping,milling, ball-milling, grinding, sprucing, or blending the plantbiomass. 18) The method of any preceding numbered embodiment, whereinthe incubating of step (b) occurs in an incubation solution comprisingwater. 19) The method of any preceding numbered embodiment, wherein theincubating of step (b) occurs at a temperature of from 15° C. to 95° C.20) The method of any preceding numbered embodiment, wherein theincubating of step (b) is conducted from 15 minutes to 1 hour. 21) Themethod of any preceding numbered embodiment, wherein thethermochemically treating of step (c) comprises heating the physicallytreated plant biomass in the acidic solution or the alkali solution. 22)The method of numbered embodiment 21, wherein the heating is at atemperature of from 50° C. to 150° C. 23) The method of numberedembodiment 21 or 22, wherein the heating is conducted from 30 minutes to4 hours. 24) The method of any preceding numbered embodiment, whereinthe incubated plant biomass is thermochemically treated in an alkalisolution having a pH from 8 to 11. 25) The method of numbered embodiment24, wherein the alkali solution comprises at least one of sodiumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,aqueous ammonia, ammonium sulfate, or ammonium hydroxide. 26) The methodof any preceding numbered embodiment, wherein the incubated plantbiomass is thermochemically treated in an acidic solution having a pHfrom 4 to 6. 27) The method of numbered embodiment 26, wherein theacidic solution comprises at least one of sulfuric acid, hydrochloricacid, nitric acid, phosphoric acid, acetic acid, maleic acid, fumaricacid, or oxalic acid. 28) The method of any preceding numberedembodiment, wherein the plant biomass comprises at least one of sugarcane, corn stover, corncob, wheat bran, wheat straw, hardwood, orsoftwood. 29) The method of any preceding numbered embodiment, whereinthe plant biomass comprises at least one of cellulose, chitin, chitosan,xylan, xyloglucan, mixed-linkage glucan, mannan, or lignocellulose. 30)The method of any preceding numbered embodiment, wherein the one or morepolysaccharide-cleaving enzymes comprises at least one of cellulase,xylanase, xyloglucanase, endo-glucanase, cellobiohydrolase, mannanase,lichenase, or lytic polysaccharide monooxygenase (LPMO). 31) The methodof any preceding numbered embodiment, wherein the one or morepolysaccharide-cleaving enzymes comprises at least one of AA9, AA10,AA11, AA13, AA14, or AA15. 32) The method of any preceding numberedembodiment, wherein the one or more of the polysaccharide-cleavingenzymes is prepared from Trichoderma reesei fungi. 33) The method of anypreceding numbered embodiment, wherein the one or morepolysaccharide-cleaving enzymes is operably linked to a catalyticmodule. 34) The method of any preceding numbered embodiment, wherein theone or more polysaccharide-cleaving enzymes is operably linked to anon-catalytic module. 35) The method of numbered embodiment 34, whereinthe non-catalytic module is a carbohydrate-binding module. 36) A methodfor producing an ingredient for human consumption, the methodcomprising: (a) pretreating a plant biomass, wherein the pretreatingcomprises: (i) physically treating the plant biomass; (ii) incubatingthe plant biomass in an incubation solution having a pH from 6.6 to 7.4to solubilize a portion of the monosaccharides and/or disaccharides andremoving a portion of the solubilized monosaccharides and/ordisaccharides; and (iii) thermochemically treating the plant biomass inone of (i) an acidic solution having a pH from 2 to 6.5 or (ii) analkali solution having a pH from 7.5 to 12; (b) contacting, in asolution or suspension, one or more polysaccharide-cleaving enzymes andthe pretreated plant biomass to form one or more oligosaccharides; and(c) isolating a portion of the one or more oligosaccharides to form theingredient.

EXAMPLES

The following illustrative examples are representative of embodiments ofthe compositions and methods described herein and are not meant to belimiting in any way.

Example 1—Exemplary Process

The following steps can be performed to generate an ingredient asprovided herein:

-   -   1. Physical pretreatment of a plant biomass: Mix 100 g of milled        corncob with water to a 10% (w/w) solids concentration and mix        for 60 minutes at room temperature. At the end of the 60 minutes        stop mixing and filter out the liquid, while keeping the solids.    -   2. Re-suspend the solids in water at a concentration of 10%        (w/w) solids in suspension. Start mixing, heat to 95° C., and        mix for 60 minutes at 95° C.    -   3. At the end of 60 minutes of heating, add 6 g of sodium        hydroxide (0.2-3% by weight of corncob) and continue stirring.        Heat to a temperature of 95° C. and mix for 60 minutes to break        down the hemicelluloses present in corncob. At the end of the 60        minutes, stop heating and cool down to a temperature of 50° C.    -   4. Hydrolysis: Add cellulolytic enzymes (e.g., from Trichoderma        reesei) and incubate at 50° C. at pH 5.5 (adjusted by 1 mol/L        sulfuric acid and/or 1 mol/L sodium hydroxide) for 72 hours.    -   5. Separation of the biomass: At the end of the hydrolysis,        separate the liquid from the products through a solid-liquid        separator.    -   6. Enzyme separation following enzymatic hydrolysis: the liquid        fraction from the slurry includes enzymes, oligosaccharides,        water, and salts that need to be separated. Use a 10 kDa hollow        fiber membrane to separate the enzyme proteins and other        macromolecules.    -   7. Salts are removed using ion-exchange columns at ≤45° C.        -   a. Cation column: Strongly acidic cation exchange resin,            cross-linked polystyrene matrix, sulfonate functional group,            Na⁺ counter-ion.        -   b. Anion column: Macroporous, weakly basic anion exchange            resin, cross-linked polystyrene matrix, dimethyl-tertiary            amine functional group, OH⁻ counter-ion.    -   8. Oligosaccharides concentration: The desired oligosaccharides        are selectively concentrated through nanofiltration at room        temperature.    -   9. Concentration: Concentrate the liquid to 40-75% at 60-80° C.    -   10. Spray drying: Spray dry with inlet temperatures of        130-160° C. and outlet temperatures of 65-85° C.

Example 2—Removal of Soluble Saccharides by a Washing Cycle (i.e.,Incubation Cycle)

The following steps were performed to remove soluble saccharides fromplant biomass:

-   -   1. Water was added to 100 mg of four plant biomass types (i.e.,        cane, wheat, cob, and willow) to a concentration of 10% (w/v)        and incubated at 45° C. for 30 minutes, after which suspensions        were centrifuged and supernatants were removed.    -   2. Step 1 was repeated 5 times.    -   3. 2.5 μl of each supernatant fraction was analyzed by        thin-layer chromatography (TLC).

The thin-layer chromatogram of FIG. 1 shows the presence of solublesaccharides washed from four types of plant biomass in the fivesequential washing cycles or incubation cycles (1, 2, 3, 4, and 5).Undesired monosaccharides and disaccharides, such as glucose, sucrose,and maltose are arrowed. The results of the TLC showed that for allplant biomasses, the supernatants removed after the first washing cycleshave abundant monosaccharides and disaccharides present in them. Thus,the washing cycle successfully removed the monosaccharides anddisaccharides from the plant biomass. Supernatants from subsequentwashing cycles have significantly fewer monosaccharides anddisaccharides present in them, if any at all, showing that the plantbiomass has minimal monosaccharides and disaccharides remaining in itafter a washing cycle as provided herein.

Example 3—Absence of Washed Soluble Saccharides in Enzyme Hydrolyses

The following steps were performed to show the absence of solublesaccharides in enzyme hydrolyses:

-   -   1. The washed cob and willow plant biomasses from Example 2 were        each incubated in 1% (w/v) NaOH at 99° C. for 30 minutes and        then cooled. 100 mg of unwashed cob and willow biomasses were        also each incubated in 1% (w/v) NaOH at 99° C. for 30 minutes        and then cooled.    -   2. 150 μl of the resulting suspensions were each mixed with 150        μM ammonium acetate (pH 5.5) and 150 μl of an enzyme composition        comprising beta xylanase and cellobiohydrolase. Suspensions were        then incubated at 50° C. for 16 hours to allow the enzyme        reactions with the plant biomasses to occur.    -   3. 2.5 μl of each supernatant fraction was analyzed by TLC.

The thin layer chromatogram of FIG. 2 shows the products of enzymedigestion of the four types of biomass that have (+) or have not (−)been washed as in Example 2. The results show that after enzymedigestion the end products included glucose, sucrose, and maltose forthe unwashed cob and willow plant biomasses. However, the end productsafter enzyme digestion did not include glucose, sucrose, and maltose forthe washed cob and willow plant biomasses.

Example 4—Adding Polymer to Oligosaccharide Solutions Enables them toDry into a Hard Glaze

The following steps were performed to show that adding polymer tooligosaccharide solutions enables them to dry into a hard glaze:

-   -   1. 100 μl of 10-320 mM (10, 20, 40, 80, 160, and 320 mM)        cellobiose±1% w/v birchwood xylan was pipetted onto a glass        plate.    -   2. Samples were dried at 37° C.    -   3. Samples were scored with a knife to test whether or not the        ingredient settled into a solid glaze. With cellobiose alone, no        firm glaze was formed, and the dried powder readily cracked when        pressure was applied with a knife. In contrast, the composition        with 1% w/v xylan added and with cellobiose at 80 mM or less the        composition dried to form a solid, off-white, translucent        surface that was strong enough to be scored with a knife leaving        an indentation but without cracking. With a cellobiose        concentration of 160 mM or higher (5.5% w/v, or 550% w/w as        compared with xylan) the morphology of the glaze reverted to        that without xylan present. That is, no firm glaze was formed,        and the dried powder readily cracked when pressure was applied        with a knife (see FIG. 3).

Example 5—Demonstration of a Composition Comprising Two Oligosaccharidesand a Polysaccharide in Food Products

The following steps were performed to demonstrate a compositioncomprising two oligosaccharides and a polysaccharide in food products:

-   -   1. 4 g birchwood xylan was dissolved in 75 ml water with        boiling.    -   2. 12 g cellobiose and 24 g xylo-oligosaccharides (primarily        degree of polymerization (DP) 2-6) was added in 3 g increments        and dissolved with boiling.    -   3. The mixture was reduced to 50 ml with heating and formed a        thick solution with the consistency and appearance of cloudy        honey but less sweet.    -   4. 10 mL of the mixture was mixed with 12 g oats to make a        flapjack/cereal bar mixture and separately 10 mL of the mixture        was also mixed with 6 g fruit and 6 g nuts to make a cereal bar        mixture.    -   5. Samples were baked at 100° C. for 10 minutes and then left to        cool and dry overnight.

As shown in FIG. 4, panel A (flapjack/cereal bar) and panel B (fruit andnut bar), the produced flapjack and fruit and nut bar were of adesirable texture and consistency in line with flapjack/cereal bars andfruit and nut bars made with known syrups typically used in baking.

For the flapjack/cereal bar, the thick solution from step 3 helped tobind together the mixture in step 4. The combined effect of theingredient with the properties of oats yielded a grainy surface textureconsistent with flapjack/cereal bars produced using conventional sugar.The ingredient created a firm, chewy, moist, viscous texture yielding amouthfeel consistent with what is expected from these types of foodproducts, but which is not present in oats alone. The product was mildlysweet and contained no bitter or off-flavors, that can be characteristicof high-intensity sweeteners.

For the fruit and nut bar, the thick solution from step 3 helped to bindtogether the mixture in step 4. It also added a smooth, shiny glazedsurface, which is a core part of the aesthetic qualities of such foodproducts, and which would not be created if the ingredient comprisedoligosaccharides alone (i.e., oligosaccharides without thepolysaccharides). The ingredient created a firm, chewy, moist, andviscous texture yielding a mouthfeel consistent with what is expectedfrom these types of food products, but which is not present in eitherthe nuts or the fruit alone. The product was mildly sweet and containedno bitter or off-flavors, that can be characteristic of high-intensitysweeteners.

Example 6—Process for Making an Ingredient

The following steps can be performed to make an ingredient as providedherein:

-   -   1. Heat 15% w/v of corncob (ground through a 1 mm pore size        filter) in 1% w/v NaOH for 1 hour at 90° C., thereby        solubilizing a portion of the polymeric components of the        biomass.    -   2. Adjust to pH 5.5 with sulfuric acid.    -   3. Extract a volume comprising only liquid components of the        reaction representing 15% of the total volume of the reaction        (“soluble polymers”); retain the remaining 85% volume of the        reaction comprising all of the insoluble biomass fraction        (“remaining biomass”).    -   4. To the remaining biomass, add a cellulolytic enzyme cocktail        (e.g., an enzyme cocktail from Trichoderma reesei, including        cellulase, xylanase, arabinofuranosidase, LPMO, etc.) to 0.5%        w/v and incubate at 50° C. for 24 hours.    -   5. Separate soluble oligomeric reaction products from undigested        insoluble polymeric compounds by filtration.    -   6. Purify oligomeric reaction products by sequentially employing        microfiltration, ultrafiltration, and ion-exchange        chromatography (e.g., cross-flow filtration on a ceramic        membrane; filtration can be performed using 110 0.45 μm cut-off        Inside Ceram candle filters (TiO2, Ø 25 mm×L 1178 mm HD 6 mm, 8        channels per membrane) supplied by TAMI industries at a feed        pressure of maximum 3 bar).    -   7. Purify the soluble polymers by employing ultrafiltration        (e.g., 10 kDa spiral wound membranes (Snyder ST-2B-6338, PES,        feed spacer thickness 31 mm) run on Alfa Laval ultrafiltration        unit).    -   8. Recombine the solutions formed in steps 6 and 7 and further        purify and concentrate by employing nanofiltration to form the        ingredient.

Example 7—Viscosity Measurements of Different Solutions

The following steps were performed to measure viscosity of differentsolutions:

-   -   1. Three saccharide solutions comprising cellobiose (Cell₂),        xylo-oligosaccharides of primarily DP 2-6 (XOS), and polymeric        beechwood xylan (BWX) were created by boiling saccharides in        water. Final concentrations were:        -   a. Sample 1: 0.33 g/ml Ce112, 0.66 g/ml XOS, 0.13 g/ml BWX        -   b. Sample 2: 0.17 g/ml Ce112, 0.33 g/ml XOS, 0.07 g/ml BWX;        -   c. Sample 3: 0.54 g/ml Ce112, 0.52 g/ml XOS, 0.07 g/ml BWX.    -   2. The samples were tested using a Brookfield HDB VE        roto-viscometer using standard testing procedures. A 400 mL        sample was taken in a tall-form beaker to ensure that no        container effects occurred. The instrument was operated as per        the manufacturer's instructions with respect to ranges:        rotoviscometry using spindle code 61, spindle speed 100 rpm, and        at 22° C.

Sample number Viscosity (cps) 1 393 2 13 3 26

Sample 1 had a consistency like that of thick honey that needed mixingin order to dilute into aqueous solutions. In contrast, Samples 2 and 3were much runnier and could be readily mixed into aqueous solutions. Theresults showed that the viscosity of the compositions was affected moreby the polysaccharide concentration than the overall concentration ofoligosaccharide and polysaccharide. Samples 2 and 3 have the samepolysaccharide concentration, but Sample 3 has twice the concentrationof total oligosaccharide and polysaccharide than Sample 2. The viscosityof Sample 3 is twice the viscosity of Sample 2, in line with a linearrelationship between the overall concentration and viscosity. However,there is an exponential increase in the viscosity values as theconcentration of the polysaccharide increases. The polysaccharideconcentration of Sample 1 is twice that of Sample 3 and theirconcentrations of total oligosaccharide and polysaccharide are the same,yet the viscosity of Sample 1 is fifteen (15) times greater than Sample3.

Example 8—Preparation of Water-Soluble Liquid Product/Ingredient

The following steps were performed to generate a water-soluble liquidproduct/ingredient (Sample 4):

-   -   1. 100 g of milled corncobs were heated in 1 L of deionized        water containing 2.5 g of sodium chlorite at 80° C. for 1.5        hours with constant agitation. The residual volume was        reconstituted to 900 mL by addition of 200 mL deionized water        containing a further 5 g of sodium chlorite and heated at 80° C.        for 1 hour with constant agitation.    -   2. The solution was filtered through a 2 mm pore size ceramic        filter funnel with vacuum, until the filtrate was clear.    -   3. Retained solids were incubated in 1 L 0.5 M sodium hydroxide        0.1% (w/v) sodium borohydride for 17 hours at 50° C. and 115 rpm        shaking.    -   4. The pH was then adjusted to 7 with concentrated sulfuric acid        and dialyzed against tap water for 24 hours in 12,000 Dalton        cut-off dialysis tubing.    -   5. The contents of the dialysis tubing were transferred to a 2-L        beaker and the insoluble fraction allowed to sediment by        gravity.    -   6. The supernatant was decanted twice and concentrated by        evaporation at 80° C. to a volume of 120 mL.    -   7. Water-soluble polymer was precipitated by centrifugation        after addition of 3 volumes of ethanol. The resulting        supernatant was discarded and the precipitate air dried to        constant weight at room temperature.    -   8. Oligosaccharides were added to a final w/w of 10% cellobiose,        75% xylo-oligosaccharides, 15% extracted water-soluble polymer,        and mixed to homogeneity in a Waring Xtreme blender on the        lowest power setting. 94 g solids were recovered from the        blender.    -   9. Unexpectedly, all 94 g solids dissolved in 60 mL water at        50° C. with mild constant agitation (μ100 rpm), indicating a        solubility of greater than 150 g/100 g. The sample generated        (e.g., at steps 8 and 9 above) is referred to as Sample 4.

Example 9—Physicochemical Properties of the Water-Soluble Liquid Productof Example 8

Flow characteristics: The flow characteristics of the water-solubleliquid product in accordance with the present disclosure (Sample 4)described in Example 8 are detailed in Table 1 along with comparisoncompositions of water, 20% w/v glucose, 40% w/v glucose, 60% w/vglucose, 80% w/v glucose, and ≥99% glycerol (Fisher G/0650/17 assupplied). The glucose solutions were made by weighing 6, 12, 18, and 24g D-glucose respectively and making up with 90° C. water to 30 mL. Theflow characteristics were measured by timing the flow rate of 5 mL, andwhere suitable, 20 mL of the liquids from a vertically stood syringe (BDPlastipak 300613) filled with 20 mL of test liquid under gravity at roomtemperature.

TABLE 1 Flow Characteristics Time for 5 mL flow Time for 20 mL flowSample (seconds) (seconds) Water 2 13 20% w/v glucose 2.3 13.5 40% w/vglucose 2.5 15 60% w/v glucose 3 20 80% w/v glucose 5 38 ≥99% Glycerol256 Not Determined Sample 4 237 Not Determined

The increased time taken for a sample to flow out of the bottom of thesyringe (i.e., has a lower flow rate) correlates with increasedviscosity of the sample. The lower the flow rate, the moresyrup-like/sticky and viscous the liquid sample is. Measured flow ratesfor Sample 4 are similar to glycerol and lower than all the glucosesolutions tested. This property of Sample 4 makes it more useful thanthe glucose solutions and water as a binder in foodstuffs, such ascereal bars, as well as providing sweetness to the product.

Color: The color of Sample 4 corresponded to No. 30 by the StandardReference Method (SRM), a method for color assessment of wort or beer aspublished in the recommended methods of the American Society of BrewingChemists (ASBC Methods of Analysis, Beer 10. Spectrophotometric ColorMethod Approved 1958, rev. 2015. American Society of Brewing Chemists,St. Paul, Minn., U.S.A). Briefly, the absorbance of a sample is measuredin a cell of path length 1 cm at a wavelength of 430 nm. The resultantabsorbance value is multiplied by 12.7 to yield the color value. Giventhe turbidity of Sample 4, absorbance could not be measured, and anassessment was made by optical comparison to the SRM No. 30 (a darkred/brown color).

Anion exchange chromatography: Analysis of Sample 4 by high-performanceanion exchange chromatography (HPAEC) was performed using a ThermoFisher Scientific DIONEX ICS-6000 system fitted with CarboPac PA200Analytical column (3×250 mm) and CarboPac PA200G Guard column (3×50 mm)and Dionex ED Electrochemical Detector. Data was acquired withChromeleon 7 software.

Eluents A (milli Q water), B (250 mM NaOH), and C (250 mM NaOH+1 MSodium Acetate) were used to produce a mobile phase with the gradientprofile shown in Table 2.

TABLE 2 Gradient Profile Time Flow (min) (ml/min) % A % B % C 0 0.5 7525 0 3 0.5 75 25 0 6 0.5 50 50 0 15 0.5 50 42.5 7.5 20 0.5 0 0 100 230.5 75 25 0 26 0.5 75 25 0

Sample for analysis was prepared by diluting Sample 4 100-fold andpassing through a 0.45 μm syringe filter and the injection volume ofanalyte was 10 μl.

HPAEC analysis (see FIG. 5) confirmed that Sample 4 is a mixture ofmonosaccharides, disaccharides, and other oligosaccharides composed ofglucose and xylose. This is in contrast to syrups typically used in thefood industry such as corn syrup and high-fructose corn syrup thatcontain primarily monosaccharides of glucose and fructose. As a result,the product in Sample 4 when used in foodstuffs is expected to havefewer calories, a lower glycemic index, and contain fiber, in contrastto corn syrup and/or high-fructose corn syrup.

Example 10—Cold-Pressed Fruit Cereal Bar

A cold-pressed fruit cereal bar was made as follows:

-   -   1. 120 g of Sample 4 from Example 8 was heated with 30 g coconut        oil, and one-quarter (¼) teaspoon of cinnamon was added.    -   2. Once foaming, the mixture was removed from the heat and 40 g        oats, 40 g dried dates, 10 g of crisped rice, and 10 g seeds        were added.    -   3. The ingredients were mixed thoroughly until all components        were coated and the mixture was transferred into a freezer bag        and into a freezer. The contents of the bag were rolled to a        thickness of 7-10 mm and chilled at 4° C. overnight before        cutting into rectangles.

The resulting product (shown in FIG. 6) was a chewy, sticky cereal bar,which was loosely set, and containing oats and crisped rice withperceivable sweetness delivered through Sample 4 and chopped dates.

Example 11—Producing the Ingredient in a Large Manufacturing Process

The following steps can be used to produce the ingredient in a largemanufacturing process:

-   -   1. Physical pretreatment of a plant biomass: Mix 100 kg of        milled corncob with water at a concentration of 15% (w/w) solids        in suspension. Start mixing and heat to 95° C. and mix for 60        min at 95° C. Add 6 kg of sodium hydroxide (0.2-3% by weight of        corncob) and continue stirring. Heat to a temperature of 95° C.        and mix for 60 minutes to release the hemicelluloses present in        corncob. At the end of the 60 minutes, cool down to 50° C. and        adjust pH to 5.5 with sulfuric acid.    -   2. Removal of portion of soluble polysaccharide: Remove of a        portion of the soluble phase corresponding to 5-30% of the total        xylan. Neutralize with sulfuric acid and concentrate and purify        by ultrafiltration. Remove any precipitated polymer.    -   3. Hydrolysis: Add cellulolytic enzymes (e.g., from Trichoderma        reesei) to the milled corncob mixture and incubate at ˜50° C.        for 12-72 hours.    -   4. Separation of the biomass: At the end of the hydrolysis,        separate the liquid from the products through a solid-liquid        separator such as a filter press or decanting centrifuge.    -   5. Enzyme separation following enzymatic hydrolysis: The liquid        fraction from the slurry contains enzymes, oligosaccharides,        water, and salts that need to be separated. Use a 3 kDa or 10        kDa hollow fiber membrane to separate the enzyme proteins and        other macromolecules.    -   6. Salts are removed using ion-exchange columns at ≤45° C.        -   a. Cation column: Strongly acidic cation exchange resin,            cross-linked polystyrene matrix, sulfonate functional group,            and Na⁺ counter-ion.        -   b. Anion column: Macroporous, weakly basic anion exchange            resin, cross-linked polystyrene matrix, dimethyl-tertiary            amine functional group, and OH⁻ counterion.    -   7. Oligosaccharides concentration: The desired oligosaccharides        are selectively concentrated through nanofiltration at room        temperature.    -   8. Concentration: Optionally concentrate the liquid to 40-75% at        60-80° C.    -   9. Recombination: Combine purified soluble polymer with        enzyme-yielded oligomers at a dry weights ratio of 5:95-20:80.    -   10. Spray drying: Spray dry the resultant solution with inlet        temperatures of 130-160° C. and outlet temperatures of 65-85° C.

Example 12—Use of the Liquid Ingredient to Manufacture an ExtrudedCereal Bar

The following steps can be performed to use a liquid ingredient asprovided herein to manufacture an extruded cereal bar:

-   -   1. A 120 kg solution comprising 10.5 kg xylan, 57 kg        xylo-oligosaccharides, and 7.5 kg cellobiose is heated with 130        kg coconut oil and transferred into a high-speed mixer. 200 kg        rolled oats and 25 kg chopped date/raisin mixture are added and        mixed thoroughly. This is pulsed through a dough mixer and set        at 20 psi through a dough feed system.    -   2. The mixture is transferred via belt and ramshorn and baked        for 20 minutes at 180° C. The product is then transferred via        oven travellator onto a biscuit cutting line and then to        variable form fill and seal packaging when cooled to <5° C.    -   3. The resulting product is a soft, sticky cereal bar that is        loosely set and full of oats. Sweetness is delivered through the        liquid solution comprising 10.5 kg xylan, 57 kg        xylo-oligosaccharides, 7.5 kg cellobiose, and chopped dates in        the bar. The liquid solution comprising 10.5 kg xylan, 57 kg        xylo-oligosaccharides, 7.5 kg cellobiose, and the coconut oil        both act as binders to hold the other ingredients in the bar        together and give the bar its structure.

Example 13—Use of the Liquid Ingredient to Manufacture an ExtrudedBreakfast Cereal

The following steps can be performed to use a liquid ingredient asprovided herein to manufacture an extruded breakfast cereal:

-   -   1. Cereal flours (about 85-75% w/v) are combined with a solution        comprising 22.5 g xylan, 30 g cellobiose, and 97.5 g        xylo-oligosaccharides per 100 g water (about 15-25% v/v), as        well as any additives such as preservatives, and vitamins and        minerals for fortification to form a dough. This is extruded        using a twin screw extruder, which cooks the product using a        combination of heat and moisture addition and/or steam and        mechanical sheer, forming the product's shape by pushing it        through a nozzle. The product is then puffed until light in        texture and golden in color and cooled.    -   2. The result is a light, crispy, shaped breakfast cereal        product. The liquid solution comprising 22.5 g xylan, 30 g        cellobiose, and 97.5 g xylo-oligosaccharides gives the product        sweetness and helps to form the structure of the dough before        extrusion.

Example 14—Use of the Liquid Ingredient to Manufacture a Tomato Ketchup

The following steps can be performed to use a liquid ingredient asprovided herein to manufacture a tomato ketchup:

-   -   1. 4 onions and 250 g celery are blended until finely chopped in        a food processor. They are fried in 5 tbsp vegetable oil on a        low heat for 5 minutes. 4 sliced cloves of garlic are added and        cooked for a further 5 minutes. 1 tsp ground coriander, 1 short        cinnamon stick, 1 tsp all spice, one-half (½) tsp ground black        pepper, and 2 tsp celery salt are added and cooked for a further        minute.    -   2. To the mixture, 2 kg ripe, chopped tomatoes, 3 tbsp tomato        puree, one-half (½) tsp chili sauce, 200 mL white wine vinegar,        and 285 mL of a solution comprising 22.5 g xylan, 30 g        cellobiose, and 97.5 g xylo-oligosaccharides per 100 g water,        are added. The mixture is brought back to the boil and left        uncovered to simmer for an hour until the tomatoes are soft. The        cinnamon stick is discarded, the sauce mixture is blended until        smooth, and then sieved.    -   3. The resulting product is a smooth, tangy tomato ketchup. The        liquid solution comprising 22.5 g xylan, 30 g cellobiose, and        97.5 g xylo-oligosaccharides sweetens the product and adds body        to the sauce, helping to thicken the sauce and bulk the sauce        out.

Example 15—HPAEC Chromatography of Saccharides in Water Post Washing ofCorncobs

Corncobs were incubated at 100 g/L in room temperature water (“Wash” inTable 3), then water was decanted. Water was added to original totalvolume and heated to 90° C. for 60 minutes (“Wetting” in Table 3) beforebeing heated at 90° C. for 60 minutes in dilute NaOH (“Pretreating” inTable 3).

HPAEC was performed on the Wash, Wetting, and Pretreating samples andsaccharide peaks were identified (see FIG. 7 for example chromatogramfrom “Wash”). As indicated in Table 3, about 2% of the corncob at thestart of the process is glucose that can be washed out and potentiallymore glucose may be washed out. It was also noted that the pH after thewashing step decreased to 4.5.

TABLE 3 Step Glucose (g/l) Wash 2.43 g/l Wetting 1.43 g/l Pretreating0.02 g/l

Example 16—Quantifying Sugars and Organic Acids

To quantify the impact of the prewashing step on the process, threeseparate batches of corncobs were treated according to the proceduresoutlined in FIG. 10A. Samples were analyzed by HPLC for saccharides(FIG. 10B) and HPLC for organic acids (FIG. 10C). The differences in thesaccharide and organic acid compositions that were isolated from thedifferent samples (Samples A-E and 5 minutes ( 1/12 hour) to 4 hours)indicate the impact of washing. The impact of the prewashing may havebeen greater had more than 150 mL of the 600 mL per wash been extracted.Accordingly, these data indicate the direction that washing can take butnot washing's limit.

As shown, glucose, fructose, and sucrose all decreased with prewashing.Fructose and glucose are largely broken down during NaOH treatment, butsucrose is resistant to NaOH treatment. Because sucrose cannot generallybe removed from other disaccharides through filtration, it can be usefulto remove it by washing. Table 4 shows a comparison of the saccharidesin Sample Ds (“No Wash” and “Double Wash”) and Table 5 shows acomparison of saccharides in the four-hour samples (“No Wash” and“Double Wash”).

TABLE 4 No Wash Double Wash Change Change (g/L) (g/L) (g/L) (%) Glucose1.888 1.001 −0.887 −47.0 Xylose 0.000 0.000 0.000 0 Xylobiose 0.0000.000 0.000 0 Cellobiose 0.000 0.000 0.000 0 Xylotriose 0.000 0.0000.000 0 Fructose 1.966 0.958 −1.008 −51.3 Sucrose 0.232 0.045 −0.188−80.6

TABLE 5 No Wash Double Wash Change Change (g/L) (g/L) (g/L) (%) Glucose2.132 1.987 −0.145 −6.8% Xylose 1.917 2.145 +0.228 +11.9% Xylobiose0.188 0.205 +0.017 +9.1% Cellobiose 0.167 0.110 −0.057 −34.1% Xylotriose0.013 0.024 +0.011 +84.6% Fructose 0.000 0.000 0 0 Sucrose 0.292 0.125−0.167 −57.19%

Prior to the start of the “caustic cook” step (e.g., thermochemicalstep), prewashing lead to a reduction of about 50% of glucose andfructose and about 80% of sucrose. At the end of the hydrolysis,prewashing lead to small differences in small sugars. Xylose-basedsugars appear to increase in concentration (as evidenced by the negativechange), while glucose and cellobiose decrease in concentration.

A large amount of the organic acids detected are not products of thewashing but rather of the pretreatment. However, the unwashed biomassappears to have a higher total loading of organic acids than thetwo-times washed material.

Acid concentration for the washed biomass also appears lower for thesteps leading to the pretreatment stage. Table 6 shows a comparison ofthe Sample Ds and Table 7 shows a comparison of the four-hour samples.

TABLE 6 No Wash Double Wash Change Change (g/L) (g/L) (g/L) (%) Oxalate0.641 0.402 −0.239 −37.3% Citrate 0.186 0.092 −0.094 −50.5% Tartrate0.016 0.013 −0.003 −18.8% Malate 0.518 0.286 −0.232 −44.8% Succinate0.063 0.002 −0.061 −96.8% Lactate 0.000 0.000 0 0 Formate 0.000 0.000 00 Acetate 0.295 0.003 −0.292 −99.0%

TABLE 7 No Wash Double Wash Change Change (g/L) (g/L) (g/L) (%) Oxalate1.776 1.355 −0.421 −23.7% Citrate 0.551 0.000 −0.551 −100.0% Tartrate0.000 0.000 0 0 Malate 0.403 0.240 −0.163 −40.4% Succinate 0.037 0.000−0.037 −100.0% Lactate 0.078 0.141 +0.063 +80.8% Formate 0.257 0.187−0.07 −27.2% Acetate 3.727 3.512 −0.215 −5.8%

Impact of washing on organic acid content, as shown in “Change”(calculated by “No Wash” minus “Double Wash”), is noticeable both at theend of the wetting stage (Sample D) and at the end of the hydrolysisreaction (4-hour Sample) (see, e.g., Tables 6 and 7). At the end of thewetting stage (Sample D), all other acids except for lactate and formate(n/d) were detected and were at a lower concentration for the materialthat has been washed twice.

At the end of the hydrolysis, acetate content is higher (hydrolysisreleases acetate). Other acids continue to be lower for the washedbiomass than the unwashed (with the exception of lactate, which ispresent in low concentrations).

Visual observation of the samples is shown in FIG. 10D. After twowashes, the corncobs released fewer colored compounds and the liquor islighter. Without being bound by any one particular theory, coloredcompounds are likely phenols and organic acids released during washing.

Example 17—Comparison of Cold-Pressed Cereal Bar

Cold-press cereal bars were prepared according to the recipe as before(see Example 10).

Soluble polysaccharides and insoluble polysaccharides were used in thecereal bars for comparison. The soluble and insoluble polysaccharideswere as follows:

-   -   Soluble polysaccharides: 60 mL water containing 94 g dry        ingredient with a composition 10% dry w/w cellobiose, 75%        xylo-oligosaccharides, and 15% extracted water-soluble polymer        (Sample 4 as described above in Example 8).    -   Insoluble polysaccharides: 60 mL water containing 94 g dry        ingredients with a composition 10% dry w/w cellobiose, 75%        xylo-oligosaccharides, and 15% micro-crystalline cellulose.

With reference to FIG. 11A, while cereal bars made with insolublepolysaccharide looked like solid bars when placed on the table, theystarted falling apart as soon as they were lifted from the table andtaken in hand due to their soft texture and the ingredients not beingbound together well. In contrast, cereal bars made with solublepolysaccharide could be handled with ease and maintained their shape.

Hardness and stickiness of the cereal bars were measured using theTA-XTPlusC Texture Analyser (Stable Microsystems, UK) using the“ExponentC” software. Sample with 9.6 cm×3.8 cm×1 cm dimensions (L×W×H)was placed centrally under the probe. A 6 mm diameter aluminiumcylindrical probe was used in a penetration test with “Return To Start”mode and 30 kg load-cell. Once the probe triggered on the surface, itpenetrated the 2 mm distance into the sample with 2 mm/s speed. At thispoint (2 mm depth), the force value was recorded and taken as a measureof “hardness” of the sample. The probe then withdrew from the sample atwhich point the maximum force to withdraw or “stickiness” was recorded.Pre-test speed was 1 mm/s and post-test speed 10 mm/s.

Results showed that the hardest bar was the one made with solubleingredient, while lower values were obtained for insoluble ingredient(FIG. 11B). Likewise, the soluble polysaccharide-containing bar hadhigher stickiness and the insoluble polysaccharide-containing bar lowerstickiness. These results confirmed visual and tactile observations.

TABLE 8 Results Sample Hardness (g) Stickiness (g) Cereal bar 169.19−20.33 with soluble polysaccharide Cereal bar 47.43 −11.24 withinsoluble polysaccharide

TABLE 9 Texture Analyzer Settings Mode: Measure Force in CompressionOption: Return To Start Pre-Test Speed: 1.0 mm/s Test Speed: 2.0 mm/sPost-Test Speed: 10.0 mm/s Distance: 2 mm Trigger Type: Auto - 20 g TareMode: Auto Data Acquisition Rate: 400 pps

Hardness of cereal bars was further measured using the TA-XTPlusCTexture Analyser (Stable Microsystems, UK) using the “ExponentC”software. Sample with 9.6 cm×3.8 cm×1 cm (L×W×H) dimensions was placedcentrally under the knife. A Knife Edge was used in a cutting test with“Return To Start” mode and 30 kg load-cell. Once the knife triggered onthe surface, it penetrated 5 mm into the sample with 2 mm/s test speed.Maximum force measured during the cutting test was recorded as hardnessof the sample. Pre-test speed was 1.5 mm/s and post-test speed was 10mm/s.

Results obtained using the cutting method confirm the results obtainedusing the penetration method described above. Results show that thehardest bar was the one made with soluble polysaccharides, while lowervalues were obtained for insoluble polysaccharide-containing bars, theinsoluble polysaccharide-containing bar being softer (FIG. 11C).

TABLE 10 Results Sample Hardness (kg) Cereal bar with 0.73 solublepolysaccharide Cereal bar with 0.34 insoluble polysaccharide

TABLE 11 Texture Analyzer Setting Mode: Measure Force in CompressionOption: Return To Start Pre-Test Speed: 1.5 mm/s Test Speed: 2.0 mm/sPost-Test Speed: 10.0 mm/s Distance: 5 mm Trigger Type: Auto - 25 g TareMode: Auto Data Acquisition Rate: 400 pps

Example 18—Viscosity of Saccharide Composition with InsolublePolysaccharide Vs. Soluble Polysaccharide

The following samples were prepared:

-   -   Sample 1: 60 mL water containing 94 g dry ingredients with a        composition 10% dry w/w cellobiose, 75% xylo-oligosaccharides,        and 15% extracted water-soluble polymer.    -   Sample 2: 60 mL water containing 94 g dry ingredient with a        composition 10% dry w/w cellobiose, 75% xylo-oligosaccharides,        and 15% micro-crystalline cellulose.

“Sample 4” of Example 8, “Soluble polysaccharides” of Example 17, and“Sample 1” of Example 18 are substantially identical and/orinterchangeable. Further, “Sample 2” of Example 18 and “Insolublepolysaccharides” of Example 17 are substantially identical and/orinterchangeable.

Sample were analyzed for flow characteristics as described in Example 9.Data confirm that soluble polysaccharides modulate the viscosity of theoligosaccharide composition (Table 12). This can enable fine-tuning ofsolution viscometric properties in a way not generally possible witholigosaccharide alone.

TABLE 12 Time for 5 mL flow Time for 20 mL flow Sample (seconds)(seconds) Water 2 13 20% w/v glucose 2.3 13.5 40% w/v glucose 2.5 15 60%w/v glucose 3 20 80% w/v glucose 5 38 ≥99% Glycerol 256 Not DeterminedOligosaccharides with 237 Not Determined soluble polysaccharide(Sample 1) Oligosaccharides with 29 58 insoluble polysaccharide

Sequence Listing LPMO AA9 LPMO from Podospora anserina (SEQ ID NO: 1).Genbank ID CAP67740 1mkgllsvaal slavsevsah yifqqlstgs tkhgvfqyir qntnynspvt dlssndlrcn 61eggasgantq tvtvragdsf tfhldtpvyh qgpvsvylsk apgsassydg sgtwfkikdw 121gptfpggqwt lagsytaqlp scitdgeyll riqslgihnp ypagtpqfyi scaqikvtgg 181gsvnpsgvai pgafkatdpg ytaniysnfn sytvpgpsvf scgsngggss pvepqpqptt 241tlvtstrapv atqpagcava kwgqcggngw tgcttcaags tcntqnayyh qcv LichenaseGH16 lichenase from Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO: 2).GenBank ID CAA86922.1 1mpylkrvlll lvtglfmslf avtatasaqt ggsffdpfng ynsgfwqkad gysngnmfnc 61twrannvsmt slgemrlalt spaynkfdcg enrsvqtygy glyevrmkpa kntgivssff 121tytgptdgtp wdeidieflg kdttkvqfny ytngagnhek ivdlgfdaan ayhtyafdwq 181pnsikwyvdg qlkhtatnqi pttpgkimmn lwngtgvdew lgsyngvnpl yahydwvryt 241 kkXylanaseGH5 arabinoxylanase from Ruminiclostridium thermocellum (SEQ ID NO: 3).GenBank ID ABN53395.1 1mgasiktsik irtvafvsii aialsilsfi pnrayaspqr grprlnaart tfvgdngqpl 61rgpytstewt aaapydqiar vkelgfnavh lyaecfdpry papgskapgy avneidkive 121rtrelglylv itignganng nhnaqwardf wkfyapryak ethvlyeihn epvawgppys 181sstanppgav dmeidvyrii rtyapetpvl lfsyavfggk ggaaealkdi rafnkavfgn 241enavwtneav afhgyagwqe ttiaveellk agypcfmtey aggawgsgmg gldveltyel 301erlgvswltf qyipptgvsd dvtkpeyfsa lvensglswt pdygnwpaar gvygngglar 361etatwinnfl tgttrieaed fdwggngvsy ydtdsvnvgg qyrpdegvdi ektsdtgggy 421nvgwisegew leytirvrnp gyynlslrva gisgsrvqvs fgnqdktgvw elpatggfqt 481wttatrqvfl gaglqklrin alsggfnlnw ielspistgt ipdgtykfln rangktlqev 541tgnnsiitad ykgiteqhwk iqhigggqyr issagrgwnw nwwmgfgtvg wwgtgsstcf 601iisptgdgyy rivlvgdgtn lqissgdpsk iegkafhgga nqqwailpvs apafptglsa 661vldssgntan ltwnaapgan synvkrstks ggpyttiatn itstnytdtg vatgtkyyyv 721vsaysngvet lnsaeailqy pkltgtvigt qgswnnignt ihkafdgdln tffdgptang 781cwlgldfgeg vrnvitqikf cprsgyeqrm iggifqgank edfsdavtlf titslpgsgt 841ltsvdvdnpt gfryvrylsp dgsngniael qffgtpagee nddvhlgdin ddgninstdl 901qmlkrhllrs irltekqlln adtnrdgrvd stdlallkry ilrvittlGH5 xylanase from Gonapodya prolifera (SEQ ID NO: 4).GenBank ID KXS18720.1 1marlsslial vlafvavsap alaargrprl ngktfvadsg vplrgpftst ewtpavpaan 61ianmrnynfn aihlyaetfd pnypaagsqk pgyaatrvdq ivaatkaanm yvvivlanga 121nngkfnlnya kdfwsfyaar yknethviye ihnepvqwgp pyisstqspg aysmnadcyk 181iiravapdtp vllftyasig ggssaagavk daqsfntavf gnanaqwtne aiaihgywga 241qgasdaakal naagfsvvlt efaaatspts pnggqdtvlt gfmeqqgvsw ltflhvpptg 301vsgdvtdpnq ytnrmtaagi gfdrdpglna vgggqaapvp vpapapvpsp vpapvpavpa 361vrtttarpap spspvpapvp apapvpapvp apvpapvpap vpapvpaspa atttrrhrtr 421pprtttapav papppaatpk vcgGH30 xylanase from Dickeya chrysanthemi (SEQ ID NO: 5).GenBank ID AAB53151.1 1mngnvslwvr hclhaalfvs atagsfsvya dtvkidanvn yqiiqgfggm sgvgwindlt 61teqintaygs gvgqiglsim rvridpdssk wniqlpsarq ayslgakima tpwsppaymk 121snnslinggr llpanysayt shlldfskym qtngaplyai siqnepdwkp dyescewsgd 181efksylksqg skfgslkviv aeslgfnpal tdpvlkdsda skyvsiiggh lygttpkpyp 241laqnagkqlw mtehyvdskq sannwtsaie vgtelnasmv snysayvwwy irrsygllte 301dgkvskrgyv msqyarfvrp galriqaten pqsnvhltay kntdgkmviv avntndsdqm 361lslnisnanv tkfekystsa slnveyggss qvdssgkatv wlnplsvttf vskGH30 xylanase from Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO: 6).GenBank ID CAA97612.1 1miprikktic vllvcftmls vmlgpgatev laasdvtvnv saekqvirgf ggmnhpawag 61dltaaqreta fgngqnqlgf silrihvden rnnwykevet aksavkhgai vfaspwnpps 121dmvetfnrng dtsakrlkyn kyaayaqhln dfvtfmknng vnlyaisvqn epdyahewtw 181wtpqeilrfm renagsinar viapesfqyl knlsdpilnd pqalanmdil gthlygtqvs 241qfpyplfkqk gagkdlwmte vyypnsdtns adrwpealdv sqhihnamve gdfqayvwwy 301irrsygpmke dgtiskrgyn mahfskfvrp gyvridatkn pnanvyvsay kgdnkvviva 361inksntgvnq nfvlqngsas nvsrwitsss snlqpgtnit vsgnhfwahl paqsvttfvv 421 nrGH30 xylanase from Bacteroides ovatus (SEQ ID NO: 7).GenBank ID SDY64378.1 1mknitllfcl flanillgac sggedekkem degkgayalf lkksitvstg esqtdvvvew 61aktsweitlg egdivksvtp tsggsntgek qytkvrvscg anstmkkrtq tihlfdktne 121ttvdllveqe ppfksvtltv dpsvkyqpvv gfggmynpki wcgdnlisas qldkmygagg 181lgysilrlmi ypnesdwsad veaakaaqan gaiifacpwd ctdaladkit vngkemkhlk 241kenyeayanh liryvtfmke kgvnlyaisv qnepdmefty wtpsevvdfv kqygariret 301gvklmspeac gmqpeytdpi innaeafaqt dilaghlyqg ftdlssgyvk nrhdyicgvy 361sriqgktwwm tehlfndgen sddsskwefl kwqyslnhlg keihmcmegy csayiywylk 421rfyglmgdtd krsptsegei tkngyimahy aqyatettri kvvtnneevc ataywdektg 481evtivllnln gasqwleipl agikkasave tnetknmevi dtglmesaeg itvllsansi 541tsvrltf XyloglucanaseGH5 xyloglucanase from Bacteroides ovatus (SEQ ID NO: 8).GenBank ID ALJ47680.1 1mekqsfsdgl fsplgikrvi fmlvllttsf iscsnsdekg gslevaqeyr nlefdargsr 61qtiqidgpae whistseswc ksshtigegk qyvnitvean dtqkertatv tvsasgapdi 121iinvkqslys vpaydeyiap dntgmrdlts mqlsalmkag vnvgntfeav ivgndgslsg 181detcwgnptp nkvlfegika agfdvvripv ayshqfedaa tykiksawmd kveaavkaal 241daglyviini hweggwlnhp vdankealde rleamwkqia lrfrdyddrl lfagtnevnn 301ddangaqpte enyrvqngfn qvfvntvrat ggrnhyrhli vqayntdvak avahftmpld 361ivqnriflec hyydpydfti mpndenfksq wgaafaggdv satgqegdie atlsslnvfi 421nnnvpviige ygptlrdqlt gealenhlks rndyieyvvk tcvknklvpl ywdagytekl 481fdrttgqphn aasiaaimkg lnGH74 xyloglucanase from Trichoderma reesei (SEQ ID NO: 9)GenBank ID AAP57752.1 1mkvsrvlalv lgavipahaa fswknvklgg gggfvpgiif hpktkgvaya rtdigglyrl 61naddswtavt dgiadnagwh nwgidavald pqddqkvyaa vgmytnswdp sngaiirssd 121rgatwsftnl pfkvggnmpg rgagerlavd pansniiyfg arsgnglwks tdggvtfskv 181ssftatgtyi pdpsdsngyn sdkqglmwvt fdstssttgg atsrifvgta dnitasvyvs 241tnagstwsav pgqpgkyfph kaklqpaeka lyltysdgtg pydgtlgsvw rydiaggtwk 301ditpvsgsdl yfgfgglgld lqkpgtlvva slnswwpdaq lfrstdsgtt wspiwawasy 361ptetyyysis tpkapwiknn fidvtsesps dglikrlgwm iesleidptd snhwlygtgm 421tifgghdltn wdtrhnvsiq sladgieefs vqdlasapgg sellaavgdd ngftfasrnd 481lgtspqtvwa tptwatstsv dyagnsvksv vrvgntagtq qvaissdgga twsidyaadt 541smnggtvays adgdtilwst assgvqrsqf qgsfasyssl pagaviasdk ktnsvfyags 601gstfyvskdt gssftrgpkl gsagtirdia ahpttagtly vstdvgifrs tdsgttfgqv 661staltntyqi algvgsgsnw nlyafgtgps garlyasgds gaswtdiqgs qgfgsidstk 721vagsgstagq vyvgtngrgv fyaqgtvggg tggtssstkq sssstssass sttlrssvvs 781ttrastvtss rtssaagptg sgvaghyaqc ggigwtgptq cvapyvcqkq ndyyyqcvCellobiohydrolaseGH7 Cel7A cellobiohydrolase from Trichoderma reesei (SEQ ID NO: 10)GenBank ID CAH10320.1 1myrklavisa flataraqsa ctlqsethpp ltwqkcssgg tctqqtgsvv idanwrwtha 61tnsstncydg ntwsstlcpd netcaknccl dgaayastyg vttsgnslsi gfvtqsaqkn 121vgarlylmas dttyqeftll gnefsfdvdv sqlpcglnga lyfvsmdadg gvskyptnta 181gakygtgycd sqcprdlkfi ngqanvegwe pssnnantgi gghgsccsem diweansise 241altphpcttv gqeicegdgc ggtysdnryg gtcdpdgcdw npyrlgntsf ygpgssftld 301ttkkltvvtq fetsgainry yvqngvtfqq pnaelgsysg nelnddycta eeaefggssf 361sdkggltqfk katsggmvlv mslwddyyan mlwldstypt netsstpgav rgscstssgv 421paqvesqspn akvtfsnikf gpigstgnps ggnppggnrg ttttrrpatt tgsspgptqs 481hygqcggigy sgptvcasgt tcqvlnpyys qclGH6 Cel6A cellobiohydrolase from Trichoderma reesei (SEQ ID NO: 11)GenBank ID AAA34210.1 1mivgilttla tlatlaasvp leerqacssv wgqcggqnws gptccasgst cvysndyysq 61clpgaassss straasttsr vspttsrsss atpppgsttt rvppvgsgta tysgnpfvgv 121tpwanayyas evsslaipsl tgamataaaa vakvpsfmwl dtldktplme qtladirtan 181knggnyagqf vvydlpdrdc aalasngeys iadggvakyk nyidtirqiv veysdirtll 241viepdslanl vtnlgtpkca naqsayleci nyavtqlnlp nvamyldagh agwlgwpanq 301dpaaqlfanv yknasspral rglatnvany ngwnitspps ytqgnavyne klyihaigpl 361lanhgwsnaf fitdqgrsgk qptgqqqwgd wcnvigtgfg irpsantgds lldsfvwvkp 421ggecdgtsds saprfdshca lpdalqpapq agawfqayfv qlltnanpsf lEndoglucanase A eg1A-Aspergillus niger GH12 (SEQ ID NO: 12) 1mklpvtlaml aatamgqtmc sqydsasspp ysvnqnlwge yqgtgsqcvy vdklsssgas 61whtewtwsgg egtvksysns gvtfnkklvs dvssiptsve wkqdntnvna dvaydlftaa 121nvdhatssgd yelmiwlary gniqpigkqi atatvggksw evwygsttqa gaeqrtysfv 181sespinsysg dinaffsylt qnqgfpassq ylinlqfgte aftggpatft vdnwtasvnAspergillus niger endo-β-1,4-glucanase GH5, CBM1 (SEQ ID NO: 13) 1mrisnlivaa saasmvsalp srqmkkrdsg fkwvgtsesg aefgsalpgt lgtdytwpet 61skiqvlrnkg mnifripflm erltpdglts sfastylsdl kstvefvtns gayavldphn 121ygrfdgsiit stsdfktwwk nvatefadnd kvifdtnney hdmeqslvld lnqaaingir 181aagattqyif vegnaytgaw dwttyndnls gltdsedkii yemhqyldsd ssgtsetcvs 241stigqerlek atewlktnnk qgivgefagg vnsvceeave gmlaymsens dvwvgaswws 301agpwwgtymy sleptdgtay stylpileky fpsgdasass sasysvaaat stastttaaf 361eqtttpatqg psatnsagev nqyyqcggin wtgptvcasp ytckvqndyy yqcvaeAspergillus niger endo-β-1,4-glucanase B GH5 (SEQ ID NO: 14) 1mkfqstllla aaagsalavp hgsghkkras vfewfgsnes gaefgtnipg vwgtdyifpd 61pstistligk gmnffrvqfm merllpdsmt gsydeeylan lttvvkavtd ggahalidph 121nygryngeli sstsdfqtfw qnlagqykdn dlvmfdtnne yydmdqdlvl nlnqaaingi 181raagasqyif vegnswtgaw twvdvndnmk nltdpedkiv yemhqyldsd gsgtsetcvs 241gtigkeritd atqwlkdnkk vgfigeyagg sndvcrsavs gmleymannt dvwkgaswwa 301agpwwgdyif sleppdgtay tgmldilety lGH30 Xylanase from Trichoderma reesei (SEQ ID NO: 15) 1mkssisvvla llghsaawsy atksqyrani kinarqtyqt migggcsgaf giacqqfgss 61glspenqqkv tqilfdenig glsivrndig sspgttilpt cpatpqdkfd yvwdgsdncq 121fnltktalky npnlyvyada wsapgcmktv gtenlggqic gvrgtdckhd wrqayadylv 181qyvrfykeeg idisllgawn epdfnpftye smlsdgyqak dflevlyptl kkafpkvdvs 241ccdatgarqe rnilyelqqa ggeryfdiat whnyqsnper pfnaggkpni qtewadgtgp 301wnstwdysgq laeglqwaly mhnafvnsdt sgythwwcaq ntngdnalir ldrdsyevsa 361rlwafaqyfr farpgsvrig atsdvenvyv tayvnkngtv aipvinaahf pydltidleg 421ikkrklseyl tdnshnvtlq srykvsgssl kvtvepramk tfwleAspergillus niger endo-β-1,4-xy1anase 1 GH11 (SEQ ID NO: 16) 1mkvtaafagl lvtafaapvp epvlvsrsag inyvqnyngn lgdftydesa gtfsmywedg 61vssdfvvglg wttgsskait ysaeysasgs ssylavygwv nypqaeyyiv edygdynpcs 121satslgtvys dgstyqvctd trtnepsitg tstftqyfsv restrtsgtv tvanhfnfwa 181qhgfgnsdfn yqvmaveaws gagsasvtis sGH5 mannanase from Trichoderma reesei (SEQ ID NO: 17) 1mmmlskslls aataasalaa vlqpvprass fvtisgtqfn idgkvgyfag tncywcsflt 61nhadvdstfs hisssglkvv rvwgfndvnt qpspgqiwfq klsatgstin tgadglqtld 121yvvqsaeqhn lkliipfvnn wsdygginay vnafggnatt wytntaaqtq yrkyvqavvs 181ryanstaifa welgneprcn gcstdvivqw atsysqyvks ldsnhlvtlg deglglstgd 241gaypytygeg tdfaknvqik sldfgtfhly pdswgtnytw gngwiqthaa aclaagkpcv 301feeygaqqnp ctneapwqtt slttrgmggd mfwqwgdtfa ngaqsnsdpy tvwynssnwq 361clvknhvdai nggtttpppv ssttttssrt sstppppggs csplygqcgg sgytgptcca 421qgtciysnyw ysqclntAspergillus niger endo-β-1,4-mannanase GH26 (SEQ ID NO: 18) 1mfaklsllsl lfssaalgas nqtlsygnid ksatpearal lkyiqlqygs hyisgqqdid 61swnwveknig vapailgsdf tyyspsavah ggkshavedv iqhagrngin alvwhwyapt 121clldtakepw ykgfyteatc fnvseavndh gngtnyklll rdidaiaaqi krldqakvpi 181lfrplhepeg gwfwwgaqgp apfkklwdil ydritryhnl hnmvwvcnta dpawypgndk 241cdiatidhyp avgdhgvaad qykklqtvtn nervlamaev gpipdpdkqa renvnwaywm 301vwsgdfiedg kqnpnqflhk vyndtrvval nwegaAspergillus niger β-mannanase GH5 (SEQ ID NO: 19) 1mklsnalltl aslalanvst alpkaspaps tsssaastsf astsglqfti dgetgyfagt 61nsywigfltd nadvdlvmgh lkssglkilr vwgfndvtsq pssgtvwyql hqdgkstint 121gadglqrldy vvssaeqhdi kliinfvnyw tdyggmsayv sayggsgetd fytsdtmqsa 181yqtyiktvve rysnssavfa welaneprcp scdtsvlynw iektskfikg ldadrmvcig 241degfglnids dgsypyqfse glnftmnlgi dtidfgtlhl ypdswgtsdd wgngwitahg 301aackaagkpc lleeygvtsn hcsvegswqk talsttgvga dlfwqygddl stgkspddgn 361tiyygtsdyq clvtdhvaai gsaAspergillus niger cellobiohydrolase A GH7 (SEQ ID NO: 20) 1mhqrallfsa lltavraqqa gtlteevhps ltwqkctseg scteqsgsvv idsnwrwths 61vndstncytg ntwdatlcpd detcaancal dgadyestyg vttdgdsltl kfvtgsnvgs 121rlylmdtsde gyqtfnllda eftfdvdvsn lpcglngaly ftamdadggv skypankaga 181kygtgycdsq cprdlkfidg qanvdgweps snndntgign hgsccpemdi weankistal 241tphpcdsseq tmcegndcgg tysddryggt cdpdgcdfnp yrmgndsfyg pgktidtgsk 301mtvvtqfitd gsgslseikr yyvqngnvia nadsnisgvt gnsittdfct aqkkafgded 361ifaehnglag isdamssmvl ilslwddyya smewldsdyp enatatdpgv argtcdsesg 421vpatvegahp dssvtfsnik fgpinstfsa saAspergillus niger cellobiohydrolase B GH7, CBM1 (SEQ ID NO: 21) 1mssfqiyraa lllsilatan aqqvgtytte thpsltwqtc tsdgscttnd gevvidanwr 61wvhstssatn cytgnewdts ictddvtcaa ncaldgatye atygvttsgs elrlnfvtqg 121ssknigsrly lmsddsnyel fkllgqeftf dvdvsnlpcg lngalyfvam dadggtseys 181gnkagakygt gycdsqcprd lkfingeanc dgwepssnnv ntgvgdhgsc caemdvwean 241sisnaftahp cdsysqtmcd gdscggtysa sgdrysgtcd pdgcdynpyr lgntdfygpg 301ltvdtnspft vvtqfitddg tssgtlteik rlyvqngevi angastyssv ngssitsafc 361esektlfgde nvfdkhggle gmgeamakgm vlvlslwddy aadmlwldsd ypvnssastp 421gvargtcstd sgvpatveae spnayvtysn ikfgpigsty ssgsssgsgs sssssstttk 481atsttlktts ttssgsssts aaqaygqcgg qgwtgpttcv sgytctyena yysqclGH3 beta-glucosidase from Trichoderma reesei (SEQ ID NO: 22) 1mryrtaaala latgpfarad shstsgasae avvppagtpw gtaydkakaa laklnlqdkv 61givsgvgwng gpcvgntspa skisypslcl qdgplgvrys tgstaftpgv qaastwdvnl 121irergqfige evkasgihvi lgpvagplgk tpqggrnweg fgvdpyltgi amgqtingiq 181svgvqatakh yilneqelnr etissnpddr tlhelytwpf adavqanvas vmcsynkvnt 241twacedqytl qtvlkdqlgf pgyvmtdwna qhttvqsans gldmsmpgtd fngnnrlwgp 301altnavnsnq vptsrvddmv trilaawylt gqdqagypsf nisrnvqgnh ktnvraiard 361givllkndan ilplkkpasi avvgsaaiig nharnspscn dkgcddgalg mgwgsgavny 421pyfvapydai ntrassqgtq vtlsntdnts sgasaargkd vaivfitads gegyitvegn 481agdrnnldpw hngnalvqav agansnvivv vhsvgaiile qilalpqvka vvwaglpsqe 541sgnalvdvlw gdvspsgklv ytiakspndy ntrivsggsd sfseglfidy khfddanitp 601ryefgyglsy tkfnysrlsv lstaksgpat gavvpggpsd lfqnvatvtv diansgqvtg 661aevaqlyity pssaprtppk qlrgfaklnl tpgqsgtatf nirrrdlsyw dtasqkwvvp 721sgsfgisvga ssrdirltst lsvaAA9 LPMO from Trichoderma reesei (SEQ ID NO: 23) 1miqklsnllv talavatgvv ghghindivi ngvwyqaydp ttfpyesnpp ivvgwtaadl 61dngfvspday qnpdiichkn atnakghasv kagdtilfqw vpvpwphpgp ivdylancng 121dcetvdkttl effkidgvgl lsggdpgtwa sdvlisnnnt wvvkipdnla pgnyvlrhei 181ialhsagqan gaqnypqcfn iaysgsgslq psgvlgtdly hatdpgvlin iytsplnyii 241pgptvvsglp tsvaqgssaa tatasatvpg ggsgptsrtt ttarttqass rpsstppatt 301sapaggptqt lygqcggsgy sgptrcappa tcstlnpyya qclnGH7 beta-gluanase (EGI) from Trichoderma reesei (SEQ ID NO: 24)GenBank: AAA34212.1 1mapsvtlplt tailaiarlv aaqqpgtstp evhpklttyk ctksggcvaq dtsvvldwny 61rwmhdanyns ctvnggvntt lcpdeatcgk ncfiegvdya asgvttsgss ltmnqympss 121sggyssvspr lylldsdgey vmlklngqel sfdvdlsalp cgengslyls qmdengganq 181yntaganygs gycdaqcpvq twrngtlnts hqgfccnemd ilegnsrana ltphsctata 241cdsagcgfnp ygsgyksyyg pgdtvdtskt ftiitqfntd ngspsgnlvs itrkyqqngv 301dipsaqpggd tisscpsasa ygglatmgka lssgmvlvfs iwndnsqymn wldsgnagpc 361sstegnpsni lannpnthvv fsnirwgdig sttnstappp ppassttfst trrssttsss 421psctqthwgq cggigysgck tctsgttcqy sndyysqclGHS beta-glucanase (EGII) from Trichoderma reesei (SEQ ID NO: 25)GenBank: ABA64553.1 1mnksvaplll aasilyggav aqqtvwgqcg gigwsgptnc apgsacstln pyyaqcipga 61ttittstrpp sgpttttrat stssstppts sgvrfagvni agfdfgcttd gtcvtskvyp 121plknftgsnn ypdgigqmqh fvnedgmtif rlpvgwqylv nnnlggnlds tsiskydqlv 181qgclslgayc ivdihnyarw nggiigqggp tnaqftslws qlaskyasqs rvwfgimnep 241hdvnintwaa tvqevvtair nagatsqfis lpgndwqsag afisdgsaaa lsqvtnpdgs 301ttnlifdvhk yldsdnsgth aecttnnidg afsplatwlr qnnrqailte tgggnvqsci 361qdmcqqiqyl nqnsdvylgy vgwgagsfds tyvltetptg sgnswtdtsl vssclark

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

1. A method for producing an ingredient for human consumption, themethod comprising: (a) subjecting a biomass to a first pretreatment toreduce an average size of the biomass to produce a pretreated biomass;(b) subjecting the pretreated biomass to a second pretreatmentcomprising: (i) incubating the pretreated biomass in an aqueous solutionto solubilize monosaccharides and/or disaccharides from the pretreatedbiomass; and (ii) removing a portion of the aqueous solution comprisingsolubilized monosaccharides and/or disaccharides to produce a secondpretreated biomass; (c) subjecting the second pretreated biomass to athird pretreatment to increase a digestibility of the biomass to producea third pretreated biomass, wherein the third pretreatment is performedafter the second pretreatment; (d) contacting, in a solution or asuspension, one or more polysaccharide-cleaving enzymes and the thirdpretreated biomass to form one or more oligosaccharides; and (e)enriching the solution or suspension to increase a concentration of theone or more oligosaccharides to form the ingredient.
 2. The method ofclaim 1, further comprising removing at least 25% vol/vol of the aqueoussolution comprising the solubilized monosaccharides and/or disaccharidesat (b)(ii).
 3. The method of claim 1, wherein (b) and/or (c) isperformed at least two or more times.
 4. The method of claim 1, whereinthe third pretreatment is a thermochemical treatment comprisingincubating the gently pre-treated biomass in an alkali solution at atemperature of 50° C. to 150° C.
 5. The method of claim 4, wherein thethermochemical treatment is conducted from 5 minutes to 72 hours.
 6. Themethod of claim 1, wherein the solution or suspension comprising thestrongly pretreated biomass after (c) comprises less than 10% w/w by dryweight monosaccharides.
 7. The method of claim 1, further comprisingpurifying the one or more oligosaccharides from the solution orsuspension.
 8. The method of claim 1, wherein the portion of thesolubilized monosaccharides and/or disaccharides removed in (b) is notcombined with the one or more oligosaccharides of (e) to form theingredient.
 9. The method of claim 1, wherein the ingredient comprisesless than 15% by dry weight monosaccharides.
 10. The method of claim 1,wherein the ingredient comprises less than 60% by dry weightdisaccharides.
 11. The method of claim 1, wherein the ingredient issubstantially free of monosaccharides.
 12. The method of claim 1,wherein the one or more oligosaccharides comprise at least one of: i) acello-oligosaccharide having a degree of polymerization (DP) of from twoto six; ii) a xylo-oligosaccharide having a DP of from two to twelve;iii) an arabinoxylo-oligosaccharide having a DP of from three tofifteen; iv) a manno-oligosaccharide having a DP of from two to twelve;v) a mixed-linkage glucan oligosaccharide having a DP of from two tofive; vi) a xyloglucan oligosaccharide having a DP of from four totwelve; or vii) a chito-oligosaccharide having a DP of from two totwelve.
 13. The method of claim 12, wherein the ingredient comprises atleast two of the oligosaccharides selected from (i) to (vii).
 14. Themethod of claim 1, wherein the ingredient comprises at least 5% w/w ofthe one or more oligosaccharides.
 15. The method of claim 12, whereinthe ingredient comprises at least one of sucrose, maltose, lactose,glucose, fructose, or galactose at a concentration less than 50% dry w/wof the total dry w/w of the one or more oligosaccharides.
 16. The methodof claim 1, wherein the monosaccharides and/or disaccharides comprise atleast one of sucrose, maltose, lactose, glucose, fructose, or galactose.17. The method of claim 1, wherein (b) further comprises solubilizingone or more organic acids.
 18. The method of claim 17, wherein the oneor more organic acids comprises at least one of oxalate, tartrate,succinate, formate, citrate, malate, lactate, or acetate.
 19. The methodof claim 18, wherein the total weight of the one or more organic acidsis greater than 10% of the total dry weight of the monosaccharidesand/or disaccharides in the portion removed in (b)(ii).
 20. The methodof claim 1, wherein the first pretreatment of (a) comprises at least oneof chipping, chopping, milling, ball-milling, grinding, sprucing, orblending the biomass.
 21. The method of claim 1, wherein the secondpretreatment of (b) occurs in an aqueous solution comprising water. 22.The method of claim 1, wherein the second pretreatment of (b) occurs ata temperature of from 5° C. to 150° C.
 23. The method of claim 22,wherein the second pretreatment of (b) is conducted from 15 minutes to 1hour.
 24. The method of claim 1, wherein (c) comprises treating thegently pretreated biomass in an alkali solution having a pH from 8 to14.
 25. The method of claim 24, wherein the alkali solution comprises atleast one of sodium hydroxide, potassium hydroxide, sodium carbonate,calcium carbonate, aqueous ammonia, ammonium sulfate, or ammoniumhydroxide.
 26. The method of claim 1, wherein the biomass is a plantbiomass comprising at least one of a grain, sugar cane, corn, wheat,rice, miscanthus, sorghum residue, switch grass, bamboo,monocotyledonous tissue, hardwood tissue, or softwood tissue.
 27. Themethod of claim 1, wherein the biomass comprises at least one ofcellulose, chitin, chitosan, xylan, xyloglucan, mixed-linkage glucan,mannan, arabinoxylo-oligosaccharides, or lignocellulose.
 28. The methodof claim 1, wherein the one or more polysaccharide-cleaving enzymescomprises at least one of cellulase, xylanase, xyloglucanase,endo-glucanase, cellobiohydrolase, mannanase, lichenase, or lyticpolysaccharide monooxygenase (LPMO).
 29. The method of claim 1, whereinthe one or more of the polysaccharide-cleaving enzymes is prepared froma filamentous fungus.
 30. The method of claim 1, further comprisingincorporating the ingredient into at least one of a baked good, achocolate, a confectionery, or a drink.